JP2021020943A - Stable dual variable domain immunoglobulin protein formulations - Google Patents

Abstract

To provide pharmaceutical protein formulations of which chemical instability and physical instability have been overcome.SOLUTION: Disclosed is an aqueous formulation comprising an aqueous stable dual variable domain immunoglobulin (AS-DVD-Ig) protein and a buffer having a molarity of about 5 to about 50 mM, where the formulation has a pH of about 4.5 to about 7.5. Also disclosed is a lyophilized formulation comprising a lyophilized-stable DVD-Ig (AS-DVD-Ig) protein, where when the formulation is reconstituted the formulation comprises about 1-100 mg/ml of the LS-DVD-Ig protein, about 10-50 mM of a buffer, a polyol, about 0.01-0.2 mg/ml of a polysorbate, and has a pH of about 5-7.SELECTED DRAWING: Figure 1

Description

Related Applications This application claims the priority benefit of US Provisional Application No. 61/721364 filed on November 1, 2012. The application also claims the priority benefit of US Provisional Application No. 61/794231 filed on March 15, 2013. The contents of both preferred applications are incorporated herein by reference.

The basic principle of pharmaceutical protein preparations is that certain instabilities, such as chemical and physical instability, must be overcome. Chemical instability often leads to modification of proteins through bond formation or cleavage. Examples of problems related to chemical instability include deamidation, racemization, hydrolysis, oxidation, beta elimination, and disulfide exchange. Physical instability does not lead to covalent changes in proteins, but they are equally problematic and difficult to overcome. Physical instability is accompanied by changes in the higher-order structure (secondary and higher) of the protein, which can result in denaturation, surface adsorption, aggregation, and / or precipitation (Manning et al. (1989) Pharma. Res. 6: 903). For therapeutic proteins, chemical and physical instability can create significant challenges in formulating proteins for delivery to patients. Aggregation is often considered the most common type of physical instability. For example, exposure to a hydrophobic interface promotes physical instability of the alignment of protein molecules at that interface, causing unfolding of the protein, maximizing exposure of hydrophobic residues to air and aggregating. To start.

Highly concentrated protein preparations, especially those in liquid form, are often desirable for therapeutic purposes as they allow for smaller doses and offer the potential for subcutaneous delivery. The development of high protein concentration formulations, however, presents many challenges. For example, high protein concentrations often result in increased protein aggregation, insolubility, and degradation (see Shire et al. (2004) J. Pharma. Sci. 93: 1390 for discussion).

To date, the majority of approved therapeutic proteins are antibodies. The development of commercially feasible antibody pharmaceuticals, however, has not been straightforward despite the fact that antibodies generally have the same structure (Wang et al. (2007) J. Pharma. Sci. .96: 1). Concentration-dependent aggregation is considered to be one of the greatest challenges in formulating antibodies (Shire et al. (2004) J. Pharm. Sci. 93: 1390).

A bivariable domain immunoglobulin (DVD-Ig (TM)) protein is a multivalent binding protein that is engineered to combine the function and specificity of two monoclonal antibodies into a single molecular entity (Wu et al., See US Pat. No. 7,612,181). Given the multivalued nature of the DVD-Ig protein, these molecules hold great promise as therapeutic agents. However, DVD-Ig proteins present new formulation challenges given their much larger size (approximately 200 kDa) and complexity compared to antibodies.

U.S. Pat. No. 7,612,181

Manning et al. (1989) Pharm. Res. 6: 903 Shire et al. (2004) J.M. Pharm. Sci. 93: 1390 Wang et al. (2007) J.M. Pharm. Sci. 96: 1

The majority of bivariable domain immunoglobulin (DVD-Ig (TM)) proteins are prone to destabilization (eg, aggregation) in aqueous formulations, whereas subsets of DVD-Ig proteins are stably formulated. can do. Some DVD-Ig proteins in the aqueous state suffer certain problems such as aggregation and / or fragmentation of DVD-Ig protein monomers. Unexpectedly, the subset of DVD-Ig protein can be stably formulated in an aqueous state even at high concentrations. Such stable DVD-Ig proteins are referred to herein as aqueous stable bivariable domain immunoglobulin proteins or "AS-DVD-Ig" proteins.

In certain embodiments, the present disclosure provides a stable aqueous formulation containing an AS-DVD-Ig protein, including a high concentration AS-DVD-Ig formulation. The AS-DVD-Ig protein remains stable during storage, eg, at a concentration of 50 mg / mL or higher (eg, at a concentration of at least 50 mg / mL (as determined by size exclusion chromatography (SEC))). It is a subpopulation of DVD-Ig proteins characterized by their ability to aggregate less than 3% after accelerated storage (40 ° C.) in the formulation). In certain embodiments, the AS-DVD-Ig protein is formulated at a concentration of at least about 50 mg / mL in histidine or citrate phosphate buffer as determined by size exclusion chromatography (SEC). When stored at about 40 ° C. for 14 days, it is characterized as a DVD-Ig protein with a loss of less than about 15% in monomeric relative percentage. In certain embodiments, the AS-DVD-Ig protein, when determined by SEC, is about 40 when formulated in histidine or citrate phosphate buffer at a concentration of at least about 50 mg / mL. After 14 days of storage at ° C, it is characterized as a DVD-Ig protein with a loss of less than about 10% in monomeric relative percentage. In certain embodiments, the AS-DVD-Ig protein, when determined by SEC, is about 40 when formulated in histidine or citrate phosphate buffer at a concentration of at least about 50 mg / mL. After 14 days of storage at ° C, it is characterized as a DVD-Ig protein with a loss of less than about 5% in monomeric relative percentage. In certain embodiments, the AS-DVD-Ig protein, when determined by SEC, is formulated at a concentration of at least 50 mg / mL in citrate phosphate buffer for 14 days at 40 ° C. Is characterized as having less than 10% aggregation after storage. In certain embodiments, the AS-DVD-Ig protein is characterized as having no more than 6% aggregation after storage at 40 ° C. for 14 days, as determined by SEC, at a concentration of at least 50 mg / mL. The AS-DVD-Ig protein is stored in citrate phosphate buffer or histidine buffer. In certain embodiments, the AS-DVD-Ig protein is characterized as having no more than 5% aggregation after storage at 40 ° C. for 14 days, as determined by SEC, at a concentration of at least 50 mg / mL. The AS-DVD-Ig protein is stored in citrate phosphate buffer or histidine buffer. In certain embodiments, the AS-DVD-Ig protein is characterized as having no more than 4% aggregation after storage at 40 ° C. for 14 days, as determined by SEC, at a concentration of at least 50 mg / mL. The AS-DVD-Ig protein is stored in citrate phosphate buffer or histidine buffer. In certain embodiments, the AS-DVD-Ig protein is characterized as having no more than 3% aggregation after storage at 40 ° C. for 14 days, as determined by SEC, at a concentration of at least 50 mg / mL. The AS-DVD-Ig protein is stored in citrate phosphate buffer or histidine buffer. In certain embodiments, the AS-DVD-Ig protein is characterized as having no more than 2% aggregation after storage at 40 ° C. for 14 days, as determined by SEC, at a concentration of at least 50 mg / mL. The AS-DVD-Ig protein is stored in citrate phosphate buffer or histidine buffer. In certain embodiments, the AS-DVD-Ig protein is characterized as having no more than 1% aggregation after storage at 40 ° C. for 14 days, as determined by SEC, at a concentration of at least 50 mg / mL. The AS-DVD-Ig protein is stored in citrate phosphate buffer or histidine buffer. In certain embodiments, the AS-DVD-Ig protein is about 10% in an aqueous formulation at about 5.5 to about 6.5 pH, at a concentration of about 100 mg / mL, after storage at about 40 ° C. for 21 days. It is characterized as a DVD-Ig protein with the following monomeric relative (rel.) Peak area changes. In other embodiments, the AS-DVD-Ig protein is about 1% in an aqueous formulation at a concentration of about 100 mg / mL at about 5.5 to about 6.5 pH and after storage at about 5 ° C. for 21 days. It is characterized as a DVD-Ig protein with the following monomeric relative (rel.) Peak area changes. Examples of AS-DVD-Ig proteins that can be included in the formulations of the present disclosure include the AS-DVD-Ig protein, which has binding specificity for IL4 and IL13, IL1α and IL1β, TNFα and IL17, and PLL4 and VEGF. These include, but are not limited to.

In certain embodiments, the formulation comprises less than about 10% aggregated AS-DVD-Ig protein. In certain embodiments, the formulation comprises less than about 9% aggregated AS-DVD-Ig protein. In certain embodiments, the formulation comprises less than about 8% aggregated AS-DVD-Ig protein. In certain embodiments, the formulation comprises less than about 7% aggregated AS-DVD-Ig protein. In certain embodiments, the formulation comprises less than about 6% aggregated AS-DVD-Ig protein. In certain embodiments, the formulation comprises less than about 5% aggregated AS-DVD-Ig protein. In certain embodiments, the formulation comprises less than about 4% aggregated AS-DVD-Ig protein. In a further embodiment, the formulation comprises less than about 3% aggregated AS-DVD-Ig protein. Aggregation may be determined by SEC analysis.

In certain embodiments, the AS-DVD-Ig protein is at a concentration of about 100 mg / mL in an aqueous formulation having a pH of about 5.0 to about 6.5, eg, about 5.5 to about 6.0. It is characterized as a DVD-Ig protein with a monomer relative (rel.) Peak area change of about 10% or less after storage at about 40 ° C. for 21 days. In certain embodiments, the AS-DVD-Ig protein is in an aqueous formulation at a pH of about 5.0 to about 6.5, such as about 5.5 to about 6.0, at a concentration of about 100 mg / mL. , Characterized as a DVD-Ig protein with a monomer relative (rel.) Peak area change of about 1% or less after storage at about 5 ° C. for 21 days.

In certain embodiments, the present disclosure provides an aqueous formulation comprising an AS-DVD-Ig protein and a buffer. For example, the present disclosure provides an aqueous formulation comprising AS-DVD-Ig protein and a buffer having a molar concentration of about 5 to about 50 mM, which formulation has a pH of about 4.5 to about 7.5, For example, it has a pH of about 5 to about 6.5. In certain embodiments, the AS-DVD-Ig protein is formulated at a concentration of at least about 50 mg / mL in histidine or citrate phosphate buffer as determined by size exclusion chromatography (SEC). When stored at about 40 degrees Celsius for 14 days, it is characterized as a DVD-Ig protein with a loss of less than about 15% in monomeric relative percentage. In certain embodiments, the AS-DVD-Ig protein, when determined by SEC, is about 40 when formulated in histidine or citrate phosphate buffer at a concentration of at least about 50 mg / mL. After 14 days of storage at degrees Celsius, it is characterized as a DVD-Ig protein with a loss of less than about 10% in monomeric relative percentage. In certain embodiments, the AS-DVD-Ig protein, when determined by SEC, is about 40 when formulated in histidine or citrate phosphate buffer at a concentration of at least about 50 mg / mL. After 14 days of storage at degrees Celsius, it is characterized as a DVD-Ig protein with a loss of less than about 5% in monomeric relative percentage. In certain embodiments, the formulation comprises about 6% or less agglutination as determined by SEC analysis. In other embodiments, the formulation comprises no more than about 5% agglutination as determined by SEC analysis. In certain embodiments, the AS-DVD-Ig protein is about 10% in an aqueous formulation at about 5.5 to about 6.5 pH, at a concentration of about 100 mg / mL, after storage at about 40 ° C. for 21 days. It is characterized as a DVD-Ig protein with the following monomeric relative (rel.) Peak area changes. In other embodiments, the AS-DVD-Ig protein is about 1% in an aqueous formulation at a concentration of about 100 mg / mL at about 5.5 to about 6.5 pH and after storage at about 5 ° C. for 21 days. It is characterized as a DVD-Ig protein with the following monomeric relative (rel.) Peak area changes. In certain embodiments, the formulation comprises about 1 to about 250 mg / mL, about 1 to 200 mg / mL, about 10 to about 230 mg / mL, about 20 to about 210 mg / mL, about 30 to about 190 mg / mL, Includes about 40-about 170 mg / mL, about 50-about 150 mg / mL, about 60-about 130 mg / mL, about 70-about 110 mg / mL, or about 80-about 105 mg / mL AS-DVD-Ig protein. In certain embodiments, the buffer used in the formulation is acetate, histidine, glycine, arginine, phosphate, citrate, or citrate / phosphate buffer. In certain embodiments, the molar concentration of the buffer is in the range of 5-50 mM, eg 10-20 mM.

In certain embodiments, the present disclosure provides an aqueous formulation comprising an AS-DVD-Ig protein, a buffer, and a surfactant. For example, the present disclosure provides an aqueous formulation comprising AS-DVD-Ig protein, a buffer having a molar concentration of about 5 to about 50 mM, and a surfactant, which formulation is about 4.5 to about 7. It has a pH of .5. In certain embodiments, the formulation comprises about 1 to about 250 mg / mL, about 10 to about 230 mg / mL, about 20 to about 210 mg / mL, about 30 to about 190 mg / mL, and about 40 to about 170 mg / mL. , About 50 to about 150 mg / mL, about 60 to about 130 mg / mL, about 70 to about 110 mg / mL, or about 80 to about 105 mg / mL of AS-DVD-Ig protein. In certain embodiments, the surfactant is polysorbate, such as polysorbate 80 or polysorbate 20. In certain embodiments, the concentration of polysorbate in the formulation is about 0.001% to about 1%, about 0.005% to about 0.05%, about 0.01% to about 0.05%, or It is about 0.1%. In certain embodiments, the concentration of polysorbate 80 or polysorbate 20 in the formulation is from about 0.005% to about 0.02%. In certain embodiments, the buffer used in the formulation is acetate, histidine, glycine, arginine, phosphate, citrate, or citrate / phosphate buffer. In certain embodiments, the molar concentration of the buffer is in the range of 5-50 mM, eg 10-20 mM.

In certain embodiments, the present disclosure comprises an aqueous formulation comprising AS-DVD-Ig protein, buffer, and polyol. For example, the present disclosure provides an aqueous formulation comprising AS-DVD-Ig protein, a buffer having a molar concentration of about 5 to about 50 mM, and a polyol, which formulation is about 4.5 to about 7.5. Has a pH of. In certain embodiments, the formulation comprises about 1 to about 250 mg / mL, about 10 to about 230 mg / mL, about 20 to about 210 mg / mL, about 30 to about 190 mg / mL, and about 40 to about 170 mg / mL. , About 50 to about 150 mg / mL, about 60 to about 130 mg / mL, about 70 to about 110 mg / mL, or about 80 to about 105 mg / mL of AS-DVD-Ig protein. In certain embodiments, the polyol is sorbitol. In certain embodiments, the concentration of sorbitol in the formulation is about 20-about 60 mg / mL, about 25-about 55 mg / mL, about 30-about 50 mg / mL, or about 35-about 45 mg / mL sorbitol. .. In certain embodiments, the polyol is sucrose. In certain embodiments, the concentration of sucrose in the formulation is from about 60 to about 100 mg / mL, from about 65 to about 95 mg / mL, from about 70 to about 90 mg / mL, or from about 75 to about 85 mg / mL. In certain embodiments, the polyol is mannitol. In certain embodiments, the concentration of mannitol in the formulation is about 10 to about 100 mg / mL, or about 20 to about 80, about 20 to about 70, about 30 to about 60, or about 30 to about 50 mg / mL. Is. In certain embodiments, the buffer used in the formulation is acetate, histidine, glycine, arginine, phosphate, citrate, or citrate / phosphate buffer. In certain embodiments, the molar concentration of the buffer is in the range of 5-50 mM, eg 10-20 mM.

In certain embodiments, the present disclosure provides an aqueous formulation comprising an AS-DVD-Ig protein, a buffer, a polyol, and a surfactant. For example, the present disclosure provides an aqueous formulation comprising AS-DVD-Ig protein, a buffer having a molar concentration of about 5 to about 50 mM, a surfactant, and a polyol, which formulation is from about 4.5 to. It has a pH of about 7.5. In certain embodiments, the formulation comprises about 1 to about 250 mg / mL, about 10 to about 230 mg / mL, about 20 to about 210 mg / mL, about 30 to about 190 mg / mL, and about 40 to about 170 mg / mL. , About 50 to about 150 mg / mL, about 60 to about 130 mg / mL, about 70 to about 110 mg / mL, or about 80 to about 105 mg / mL of AS-DVD-Ig. In certain embodiments, the polyol is sorbitol. In certain embodiments, the sorbitol concentration in the formulation is from about 20 to about 60 mg / mL, from about 25 to about 55 mg / mL, from about 30 to about 50 mg / mL, or from about 35 to about 45 mg / mL. In certain embodiments, the polyol is sucrose. In certain embodiments, the concentration of sucrose in the formulation is from about 60 to about 100 mg / mL, from about 65 to about 95 mg / mL, from about 70 to about 90 mg / mL, or from about 75 to about 85 mg / mL. In certain embodiments, the polyol is mannitol. In certain embodiments, the concentration of mannitol in the formulation is about 10 to about 100 mg / mL, or about 20 to about 80, about 20 to about 70, about 30 to about 60, or about 30 to about 50 mg / mL. Is. In certain embodiments, the surfactant is polysorbate, such as polysorbate 80 or polysorbate 20. In certain embodiments, the concentration of polysorbate in the formulation is about 0.001% to about 1%, about 0.005% to about 0.05%, about 0.01% to about 0.05%, or It is about 0.1%. In certain embodiments, the concentration of polysorbate 80 or polysorbate 20 in the formulation is from about 0.005% to about 0.02%. In certain embodiments, the buffer used in the formulation is acetate, histidine, glycine, arginine, phosphate, citrate, or citrate / phosphate buffer. In certain embodiments, the molar concentration of the buffer is in the range of 5-50 mM, eg 10-20 mM.

In certain embodiments, the present disclosure discloses AS-DVD-Ig proteins, polyols (eg, sorbitol, mannitol, or sucrose), buffers (eg, acetates, histidine, glycine, arginine, phosphates, citric acid). Provided is a formulation comprising a salt, or citrate / phosphate), and a surfactant (eg, polysorbate), the formulation having a pH of about 5 to about 7, the AS-DVD-Ig. When the protein is formulated by SEC, in histidine or citrate phosphate buffer at a concentration of about 60 mg / mL, after 14 days of storage at about 40 degrees Celsius, monomeric relative It is characterized as a DVD-Ig protein with a loss of less than about 15% in percentage. In certain embodiments, the polysorbate is polysorbate 80 or polysorbate 20. In certain embodiments, the concentration of polysorbate 80 or polysorbate 20 is from about 0.005% to about 0.02%.

In certain embodiments, the present disclosure provides a formulation comprising AS-DVD-Ig protein, polyol, histidine buffer, and polysorbate, wherein the formulation has a pH of about 5 to about 7. The AS-DVD-Ig protein, as determined by SEC, is characterized as having no more than 6% aggregation after storage at 40 ° C. for 14 days, where the AS-DVD-Ig protein is phosphorus citrate. It is formulated at a concentration of at least 60 mg / mL in phosphate buffer or histidine buffer.

In certain embodiments, the present disclosure provides a formulation comprising AS-DVD-Ig protein and a buffer having a molar concentration of about 5 to about 50 mM, wherein the AS-DVD-Ig protein is determined by SEC. When formulated in histidine or citrate phosphate buffer at a concentration of about 60 mg / mL, after storage at about 40 degrees Celsius for 14 days, the monomer relative percentage is less than about 10%. Characterized as a DVD-Ig protein with a loss of, this formulation has a pH of 4.5-7.5. In certain embodiments, the formulation further comprises a surfactant, a polyol, or a combination thereof.

The present disclosure also states that, in part, the majority of DVD-Ig (TM) proteins are prone to destabilization in the lyophilized state, but subsets of the DVD-Ig protein are stably formulated in the lyophilized form. It is also based on the surprising discovery that it is possible. Such stable DVD-Ig proteins are referred to herein as lyophilized, stable bivariable domain immunoglobulin proteins or "LS-DVD-Ig" proteins. Formulations in which the DVD-Ig protein has been lyophilized suffer from certain problems, such as aggregation and / or fragmentation of the DVD-Ig protein monomer. Unexpectedly, the subset of DVD-Ig protein can be stably formulated in a lyophilized state even at high concentrations.

In certain embodiments, the present disclosure is a lyophilized formulation comprising a lyophilized and stable DVD immunoglobulin (LS-DVD-Ig) protein, which, when reconstituted, is about. It contains 1 to about 100 mg / mL LS-DVD-Ig protein, about 10 to about 50 mM buffer, polyol, about 0.01 to about 0.2 mg / mL polysolvate, about 5 to about 7, eg, about. It has a pH of 5.5 to about 6.5. In certain embodiments, the LS-DVD-Ig protein, when formulated at concentrations above about 100 mg / mL, is in an aqueous formulation at a pH of about 5.5 to about 6.5 and at about 40 ° C. After 21 days of accelerated storage, a monomer relative (rel.) Peak area change of> 10% is observed.

In certain embodiments, the LS-DVD-Ig protein, when formulated at a concentration of about 100 mg / mL or higher, has a pH of about 5.0 to about 6.5, eg, about 5. Over 10% monomeric relative (rel.) Peak area changes are observed at 5 to about 6.0 after 21 days of accelerated storage at about 40 ° C.

In certain embodiments, the present disclosure discloses buffers, surfactants, and polyols having molar concentrations of about 5 to about 50 mM (eg, histidine, sucrose, or citrate and / or phosphate). Provided is a lyophilized formulation prepared by lyophilizing an aqueous formulation, including (eg, mannitol, sorbitol, sucrose, or trehalose), which formulation is about 4.5 to about 7.5, eg, for example. It has a pH of about 5.5 to about 6.5.

In certain embodiments, the present disclosure provides an LS-DVD-Ig protein or an AS-DVD-Ig protein that is stable in a formulation having a pH of about 4.5 to about 7.5. In certain embodiments, the formulation has a pH of 5 to 6.5. In certain embodiments, the formulation has a pH of about 5.7 to about 6.3. In certain embodiments, the formulations of the present disclosure have a pH of about 5.5 to about 6.5. In certain embodiments, the formulations of the present disclosure have a pH of 5.8 to about 6.2, or a pH of 6.

In certain embodiments, the present disclosure provides a formulation comprising AS-DVD-Ig protein or LS-DVD-Ig protein, polyol, histidine buffer, and polysorbate, wherein the formulation is about 5 to about 7. The AS-DVD-Ig protein or LS-DVD-Ig protein, as determined by SEC, is characterized as having no more than 15% aggregation after 14 days storage at 40 ° C. Here, AS-DVD-Ig protein or LS-DVD-Ig protein is formulated in citrate phosphate buffer or histidine buffer at a concentration of at least 60 mg / mL. Such formulations, as determined by SEC, are lyophilized as AS-DVD-Ig protein or LS-DVD-Ig protein, which is identified as having no more than 15% aggregation after storage at 40 ° C. for 14 days. Alternatively, it may be in an aqueous state, wherein the AS-DVD-Ig protein or LS-DVD-Ig protein is formulated in citrate phosphate buffer or histidine buffer at a concentration of at least 60 mg / mL. Be made.

One advantage of the compositions of the present disclosure is that the AS-DVD-Ig protein or LS-DVD-Ig protein can be stably formulated in high concentration liquid form. In certain embodiments, the AS-DVD-Ig protein or LS-DVD-Ig protein has a concentration of about 1 to about 200 mg / mL. In certain embodiments, the AS-DVD-Ig protein or LS-DVD-Ig protein has a concentration of about 20 to about 100 mg / mL. In certain embodiments, the formulation comprises about 1 to about 250 mg / mL, about 10 to about 230 mg / mL, about 20 to about 210 mg / mL, about 30 to about 190 mg / mL, and about 40 to about 170 mg / mL. , About 50 to about 150 mg / mL, about 60 to about 130 mg / mL, about 70 to about 110 mg / mL, or about 80 to about 105 mg / mL AS-DVD-Ig protein or LS-DVD-Ig.

Examples of buffers that can be used in the formulations of the present disclosure include, but are not limited to, acetates, histidines, glycines, arginines, phosphates, and citrates. In certain embodiments, the molar concentration of buffer in the formulation is from about 5 to about 50 mM. In certain embodiments, the molar concentration of buffer is from about 10 mM to about 20 mM.

Examples of polyols that can be used in the formulations of the present disclosure include, but are not limited to, sorbitol, mannitol, and sucrose. In certain embodiments, the polyol is sorbitol. In certain embodiments, about 30 to about 50 mg / mL sorbitol is used in the formulation. In certain embodiments, the polyol is sucrose. In certain embodiments, about 70-about 90 mg / mL sucrose is used in the formulation. In a further embodiment, the polyol is mannitol. In certain embodiments, about 30 to about 50 mg / mL mannitol is used in the formulation.

Examples of surfactants that can be used in the formulations of the present disclosure include, but are not limited to, polysorbates and poloxamers. In certain embodiments, the surfactant is polysorbate, examples of which are polysorbate 80 and polysorbate 20. Other examples include poloxamer Pluronic F-68, albumin, lecithin, and cyclodextrin. In certain embodiments, the polysorbate has a concentration of about 0.05 mg / mL to about 2 mg / mL. In a further embodiment, the polysorbate has a concentration of about 0.01 to about 0.2 mg / mL.

In certain embodiments, the AS-DVD-Ig protein or LS-DVD-Ig protein comprises first and second polypeptide chains, each independently of VD1- (X1) n-VD2-C-. In the formula, VD1 is the first variable domain, VD2 is the second variable domain, C is the constant domain, and X1 is the linker, except that (X2) n is included. Given that it is not CH1, X2 is the Fc region, n is 0 or 1, and the VD1 domain on this first and second polypeptide chain is the first functional target binding. The site is formed and the VD2 domain on the first and second polypeptide chains forms a second functional target binding site. In a further embodiment, the first polypeptide chain comprises a first VD1- (X1) n-VD2-C- (X2) n, where VD1 is the first heavy chain variable domain in the formula. VD2 is the second heavy chain variable domain, C is the heavy chain constant domain, X1 is the linker, provided that it is not CH1 and X2 is the Fc region. n is 0 or 1, the second polypeptide chain comprises a second VD1- (X1) n-VD2-C- (X2) n, where VD1 is the first light chain variable in the formula. A domain, VD2 is a second light chain variable domain, C is a light chain constant domain, X1 is a linker, provided that it is not CH1 and X2 is an Fc. Not including a region, n is 0 or 1, and the VD1 domain on the first and second polypeptide chains forms a first functional target binding site, the first and second poly. The VD2 domain on the peptide chain forms a second functional target binding site. In certain embodiments, two first polypeptide chains and two second polypeptide chains, wherein the binding protein comprises four functional target binding sites. In certain embodiments, X1 is not CL.

In certain embodiments, the AS-DVD-Ig protein or LS-DVD-Ig protein comprises a polypeptide chain comprising VD1- (X1) n-VD2-C- (X2) n, where VD1 is in the formula. , 1st variable domain, VD2 is the 2nd variable domain, C is the constant domain, X1 represents an amino acid or polypeptide, X2 represents the Fc region, n is 0. Or 1.

In certain embodiments, the AS-DVD-Ig protein or LS-DVD-Ig protein used in the compositions and methods of the present disclosure comprises four polypeptide chains, of which two polypeptide chains. It comprises VD1- (X1) n-VD2-C- (X2) n, where VD1 is the first heavy chain variable domain, VD2 is the second heavy chain variable domain, and C is. It is a heavy chain constant domain, where X1 is a linker, provided that it is not CH1, X2 is an Fc region and the two polypeptide chains are VD1- (X1) n-VD2- Containing C- (X2) n, in the formula, VD1 is the first light chain variable domain, VD2 is the second light chain variable domain, C is the light chain constant domain, and X1 is. , Which is a linker, provided that it is not CH1 and X2 does not contain an Fc region, n is 0 or 1, and the four polypeptide chains of the binding protein are four. It forms a functional antigen binding site.

In certain embodiments, the AS-DVD-Ig protein or LS-DVD-Ig protein comprises a polypeptide chain, which polypeptide chain comprises VD1- (X1) n-VD2-C- (X2) n. In the formula, VD1 is the first heavy chain variable domain, VD2 is the second heavy chain variable domain, C is the heavy chain constant domain, and X1 is the linker, but , X2 is the Fc region and n is 0 or 1, provided it is not CH1. In certain embodiments, the AS-DVD-Ig protein or LS-DVD-Ig protein comprises a polypeptide chain, which polypeptide chain comprises VD1- (X1) n-VD2-C- (X2) n. In the formula, VD1 is the first light chain variable domain, VD2 is the second light chain variable domain, C is the light chain constant domain, and X1 is the linker, but , X2 does not contain the Fc region, and n is 0 or 1, provided that it is neither CH1 nor CL. In a further embodiment, (X1) n on the heavy and / or light chain is (X1) 0 and / or (X2) n on the heavy and / or light chain is (X2) 0. is there.

In certain embodiments, the AS-DVD-Ig protein or LS-DVD-Ig protein comprises first and second polypeptide chains, of which the first polypeptide chain is the first VD1-(. X1) contains n-VD2-C- (X2) n, where VD1 is the first heavy chain variable domain, VD2 is the second heavy chain variable domain, and C is the heavy chain constant. The domain, X1 is the first linker, provided that it is not CH2, X2 is the Fc region, n is 0 or 1, and the second polypeptide chain is. , 2nd VD1- (X1) n-VD2-C- (X2) n, in the formula VD1 is the first light chain variable domain and VD2 is the second light chain variable domain. , C is the light chain constant domain, X1 is the second linker, provided that it is neither CH1 nor CL, X2 does not contain the Fc region and n is 0 or It is 1.

In certain embodiments, VD1 of the first polypeptide chain and VD1 of the second polypeptide chain are derived from different first and second parent antibodies, or their binding moieties, respectively. In certain embodiments, the VD2 of the first polypeptide chain and the VD2 of the second polypeptide chain are derived from different first and second parent antibodies, or their binding moieties, respectively. In certain embodiments, the first and second parent antibodies bind to different epitopes on the same target or different targets. In certain embodiments, the first parent antibody or binding moiety thereof binds to the first target with a different potency than the potency of the second parent antibody or binding moiety thereof binding to the second target. In certain embodiments, the first parent antibody or binding moiety thereof binds to the first target with an affinity different from the affinity by which the second parent antibody or binding moiety thereof binds to the second target. To do.

In certain embodiments, the AS-DVD-Ig protein or LS-DVD-Ig protein comprises two first polypeptide chains and two second polypeptide chains.

In certain embodiments, the AS-DVD-Ig protein or LS-DVD-Ig protein comprises first and second polypeptide chains, each independently of VD1- (X1) n-VD2-C-. In the formula, VD1 is the first variable domain, VD2 is the second variable domain, C is the constant domain, and X1 is the linker, except that (X2) n is included. Given that it is not CH1, X2 is the Fc region, n is 0 or 1, and the VD1 domain on this first and second polypeptide chain is the first functional target binding. The site is formed and the VD2 domain on the first and second polypeptide chains forms a second functional target binding site. In a further embodiment, the first polypeptide chain comprises a first VD1- (X1) n-VD2-C- (X2) n, where VD1 is the first heavy chain variable domain in the formula. VD2 is the second heavy chain variable domain, C is the heavy chain constant domain, X1 is the linker, provided that it is not CH1 and X2 is the Fc region. n is 0 or 1, the second polypeptide chain comprises a second VD1- (X1) n-VD2-C- (X2) n, where VD1 is the first light chain variable in the formula. A domain, VD2 is a second light chain variable domain, C is a light chain constant domain, X1 is a linker, provided that it is not CH1 and X2 is an Fc. Not including a region, n is 0 or 1, and the VD1 domain on the first and second polypeptide chains forms a first functional target binding site, the first and second poly. The VD2 domain on the peptide chain forms a second functional target binding site.

In certain embodiments, the formulations of the present disclosure comprise a DVD-Ig protein comprising the heavy or light chain amino acid sequences set forth in Table 58 or 65.

In certain embodiments, the formulations of the present disclosure comprise a DVD-Ig protein comprising the heavy or light chain variable region amino acid sequences set forth in Table 58 or 65 (SEQ ID NOs: 28-75). As an alternative, the formulations of the present disclosure include DVD-Ig proteins, including the CDRs set forth in the heavy or light chain variable region amino acid sequences set forth in Table 58 or 65 (SEQ ID NOs: 28-75).

In certain embodiments, the formulations of the present disclosure comprise an anti-TNF / IL-17 DVD-Ig protein. In certain embodiments, the anti-TNF / IL-17 DVD-Ig protein comprises heavy and light chain sequences having the amino acid sequences set forth in SEQ ID NOs: 62 and 63, respectively.

In certain embodiments, the formulations of the present disclosure include anti-IL1α / IL1β DVD-Ig. In certain embodiments, the anti-IL1α / IL1β DVD-Ig protein comprises heavy and light chain sequences having the amino acid sequences set forth in SEQ ID NOs: 66 and 67, respectively.

In certain embodiments, the DVD-Ig protein used in the formulations of the present disclosure binds to one of the following target combinations (in any target order): CD20 / CD80, VEGF / Her2. , TNF / RANKL, TNF / DKK, CD20 / RANKL, DLL4 / PLGF, TNF / SOST (S2), IL-9 (S2) / IgE, IL-12 / IL-18, TNF / IL-17, TNF / PGE2 , IL1α / IL1β, or PLL4 / VEGF.

In certain embodiments, the formulations of the present disclosure are pharmaceutical formulations, including an aqueous pharmaceutical formulation or a lyophilized pharmaceutical formulation.

Also included in the present disclosure are methods of making and using AS-DVD-Ig protein or LS-DVD-Ig protein preparations.

In certain embodiments, the formulations of the present disclosure are used to treat disorders in a subject.

A further embodiment of the disclosure is a method of identifying either the AS-DVD-Ig protein or the LS-DVD-Ig protein. Such a method is an accelerated storage (eg, about 40 degrees Celsius) of a liquid formulation containing a DVD-Ig protein, a citrate / phosphate buffer, and a high concentration of DVD-Ig protein (eg, about 50 mg / mL or higher). Includes agglomeration tests (eg, by SEC analysis) after 14 days in degrees.

FIG. 5 is a graph comparing the DSC profile of the IgG1 antibody (briaquinumab) with the DSC profile of the DVD-Ig protein (TNF / PGE2; DVD-B), each showing a difference of 3 to 4 domain unfolding. The sample composition of the DVD-Ig solution used was 1 mg / mL DVD-Ig protein, 1 mM ionic strength histidine (pH 6), and a scanning rate of 1 ° C./min. The graph of the serum stability of various DVD-Ig proteins is shown. The domain orientation concept for different variable domain combinations is shown. The graph of the correlation between the pharmacokinetic parameters of different DVD-Ig proteins and the formation of high molecular weight (HMW) aggregates is shown. Provides a thermodynamic comparison of 14 monoclonal antibodies and 16 DVD-Ig proteins showing the melting temperature of (y-axis) molecules relative to the number of molecules at each temperature. The average starting melting temperature of the antibody was 59.2 ° C ± 3.4 ° C. The average starting melting temperature of the DVD-Ig protein was 53.6 ° C. ± 3.6 ° C. The molar ellipticity of the DVD-Ig protein as measured using a near-ultraviolet-CD scan with a wavelength of 250-320 nm is graphed.

I. Definition The term "multivalent binding protein" is used to refer to a binding protein that contains two or more target binding sites. Multivalent binding proteins may be engineered to have three or more antigen binding sites and are generally not naturally occurring antibodies.

The term "multispecific binding protein" refers to a binding protein capable of binding to two or more related or unrelated targets. An example of a multivalent binding protein is a bivariable domain (DVD) binding protein such as DVD-Ig (TM). In certain embodiments, the DVD binding protein comprises two or more antigen binding sites and is a tetravalent or multivalent binding protein. DVDs may be unispecific, i.e., capable of binding to one target, or multispecific, i.e., capable of binding to more than one target.

The term "double variable domain immunoglobulin" or "DVD-Ig (TM)" or "DVD-Ig protein" refers to a DVD-binding protein comprising two heavy chain DVD polypeptides and two light chain DVD polypeptides. Point to. Each half of the DVD-Ig contains a heavy chain DVD polypeptide and a light chain DVD polypeptide, as well as two target binding sites. Each binding site contains a heavy and light chain variable domain, with a total of 6 CDRs involved in target binding. Each variable domain (VD) in the DVD-Ig protein may be obtained from one or more "parent" monoclonal antibodies (mAbs) that bind to one or more desired antigens or epitopes. In certain embodiments, the resulting DVD-Ig molecule retains the activity of both parent mAbs. The term "DVD-Ig protein" embraces the terms AS-DVD-Ig protein and LS-DVD-Ig protein described below.

The terms "aqueous stable bivariable domain immunoglobulin" or "AS-DVD-Ig" or "AS-DVD-Ig protein" are about 1 to about 100 mg / mL (eg, about 1 to 10 mg / mL or). Concentrations in the range of about 50-100 mg / mL) and pH of about 5.0 to about 6.5, such as about 5.5 to about 6.0, given temperature and time frame, such as 5 ° C. Alternatively, it refers to a subset of DVD-Ig protein with low or low change in monomer content aggregation due to physical degradation after a stability test at 40 ° C. for 14-21 days. Different stability tests may be used to define AS-DVD-Ig. In certain embodiments, the AS-DVD-Ig protein, when determined by SEC, is in a buffered aqueous formulation at a concentration of about 50 mg / mL to about 100 mg / mL and a pH of about 5.0 to about 6. 5, eg, about 5.5 to about 6.0, defined as a DVD-Ig protein having a monomer relative peak area change of about 1% or less after storage at about 5 ° C. for 21 days. In certain embodiments, the AS-DVD-Ig protein, as determined by SEC analysis, is in a buffered aqueous formulation at a concentration of about 50 mg / mL to about 100 mg / mL and a pH of about 5.0 to about 6. 5. For example, at about 5.5 to about 6.0, it has a monomer relative (rel.) Peak area change of 10% or less after 21 days of accelerated storage at about 40 ° C. In certain embodiments, the stability test for AS-DVD-Ig protein is a citrate phosphate buffer solution at a pH of 5.0-6.5, eg, about 5.5-about 6.0. Alternatively, it may be done by testing the stability of a solution having a DVD-Ig protein concentration of 50-100 mg / mL in histidine buffer, eg, loss of monomer in that solution.

The term "aqueous formulation" refers to a liquid solution in which the solvent is water. In certain embodiments, the term "aqueous formulation" means that the solvent was water and the formulation was not lyophilized in advance (ie, did not result from the reconstruction of the lyophilized formulation). Refers to liquid preparations.

The terms "freeze-dried stable bivariable domain immunoglobulin" or "LS-DVD-Ig" or "LS-DVD-Ig protein" have low or altered monomer aggregation in the liquid state (ie, simply). Refers to a subset of DVD-Ig proteins with high levels of (mass loss). Different stability tests may be used to define LS-DVD-Ig. In certain embodiments, the LS-DVD-Ig protein is in an aqueous formulation at a concentration of about 50-100 mg / mL and has a pH of about 5.0 to about 6.5, such as about 5.5 to about 6. After 21 days of accelerated storage, eg, at about 40 ° C., when formulated at 0, greater than 10% monomeric relative (rel.) Peak area changes are observed. DVD-Ig proteins may be tested in aqueous formulations containing citrate and phosphate buffers, or histidine buffers. Monomer changes can be determined according to methods known in the art, including but not limited to SEC. In certain embodiments, accelerated storage conditions include storing the DVD-Ig protein at about 40 ° C. in the absence of light. In certain embodiments, the LS-DVD-Ig protein has a monomer relative (rel.) Peak area change of 50% or less after 14 days of accelerated storage at about 40 ° C. as determined by SEC analysis. Here, the LS-DVD-Ig protein is formulated at a concentration of at least 50 mg / mL in citrate phosphate buffer in an aqueous formulation. In certain embodiments, stability tests on the LS-DVD-Ig protein have shown that citrate phosphate at a pH of about 5.0 to about 6.5, eg, about 5.5 to about 6.0. It is done by testing the stability of a solution having a DVD-Ig protein concentration of about 50 mg / mL to 100 mg / mL in salt buffer or histidine buffer, eg, loss of monomer in that solution. May be good.

The term "pharmaceutical formulation" refers to a preparation that is in a form that allows the biological activity of the active ingredient to be effective and can therefore be administered to the subject for therapeutic use. In certain embodiments, the present disclosure provides an aqueous pharmaceutical formulation comprising AS-DVD-Ig. In another embodiment, the present disclosure provides a lyophilized pharmaceutical formulation comprising LS-DVD-Ig.

A "stable" formulation is one in which the DVD-Ig protein in the formulation essentially retains its physical and / or chemical stability and / or biological activity upon storage. Various analytical techniques for measuring protein stability are available in the art, eg, Peptide and Protein Drug Delivery, pp. 247-301, Vincent Lee Ed. , Marcel Dekker, Inc. , New York, N.K. Y. , Pubs. (1991) and Jones (1993) Adv. Drug Delivery Rev. It is outlined at 10: 29-90. In certain embodiments, the stability of the DVD-Ig protein is determined by the low percentage of degraded (eg, fragmented) and / or aggregated protein according to the percentage of monomeric protein in solution. For example, an aqueous formulation containing a stable DVD-Ig protein may contain at least 95% monomeric DVD-Ig protein, such as AS-DVD-Ig protein. Alternatively, the aqueous formulations of the present disclosure may comprise up to 5% aggregated and / or degraded DVD-Ig protein, such as AS-DVD-Ig protein.

The DVD-Ig protein substantially shows signs of aggregation, precipitation, and / or denaturation when it is measured by visual inspection of color and / or clarity, or by UV light scattering or size exclusion chromatography. If not indicated, "maintain its physical stability" in the pharmaceutical formulation. In certain embodiments of the present disclosure, stable aqueous formulations are formulations with less than about 10% aggregation and less than about 5% AS-DVD-Ig protein aggregation in the formulation.

A DVD-Ig protein is "that Retains chemical stability. " Chemical stability can be assessed by detecting and quantifying the chemically altered morphology of DVD-Ig. Chemical changes can be accompanied by resizing (eg, truncation), which includes, for example, size exclusion chromatography, SDS-PAGE and / or matrix-assisted laser desorption / ionization / time-of-flight mass spectrometry (MALDI / TOF MS). ) Can be used for evaluation. Other types of chemical changes include charge changes (eg, resulting from deamidation), which can be evaluated, for example, by ion exchange chromatography.

A DVD-Ig protein "retains its biological activity" in a pharmaceutical product if the protein in the pharmaceutical product is biologically active for its intended purpose. For example, the biological activity of a DVD-Ig protein is about the biological activity indicated at the time the pharmaceutical formulation was prepared (eg, when the biological activity of the DVD-Ig protein in a pharmaceutical formulation is determined in an antigen binding assay). If it is within 30%, about 20%, or about 10% (within assay error), it is retained.

As used herein, the term "surfactant" refers to organic substances with amphipathic structures, that is, they are groups with opposite soluble tendencies, typically oil-soluble hydrocarbons. Consists of chains and water-soluble ionic groups. Surfactants can be classified into anionic, cationic, and nonionic surfactants according to the charge of the surface active moiety. Surfactants are often used as wetting agents, emulsifying agents, solubilizing agents, and dispersing agents for various pharmaceutical compositions and preparations of biomaterials. Examples of suitable surfactants are polysorbates such as sodium lauryl sulfate, polyoxyethylene sorbitan monooleate, monolaurate, monopalmitate, monostearate, or another ester of polyoxyethylene sorbitan (eg, Tween). Commercially available Tweens (TM) (ICI Speciality Chemicals) such as (TM) 20 and Tween (TM) 80), sodium dioctyl sulfosuccinate (DOSS), lecithin, stearyl alcohol, cetostearyl alcohol, cholesterol, polyoxyethylene lysine oil, Polyoxyethylene fatty acid glycerides, poloxamers (eg, Pluronics F68 (TM) and F108 (TM), which are block copolymers of ethylene oxide and propylene oxide); include polyoxyethylene castor oil derivatives or mixtures thereof. However, it is not limited to these. In certain embodiments, the formulations of the present disclosure include polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80.

The term "tonicity modifier" or "isotonic agent" refers to a compound that can be used to adjust the tonicity of a liquid formulation. Examples of tension modifiers include glycerin, lactose, mannitol, dextrose, sodium chloride, magnesium sulphate, magnesium sulphate, sodium sulphate, sorbitol, trehalose, sucrose, raffinose, maltose, and others known to those of skill in the art. Be done.

The term "polypoly" refers to substances having multiple hydroxyl groups and includes sugars (reducing and non-reducing sugars), sugar alcohols, and sugar acids. In certain embodiments, the polyol has a molecular weight of less than about 600 kD (eg, in the range of about 120 to about 400 kD). A "reducing sugar" is a sugar that contains a free aldehyde or ketone group and is capable of reducing metal ions or covalently reacting with lysine and other amino groups in proteins. A "non-reducing sugar" is a sugar that lacks a free aldehyde or ketone group and is not oxidized by a mild oxidizing agent such as Fehling's solution or Benedict's solution. Examples of reducing sugars are fructose, mannose, maltose, lactose, arabinose, xylose, ribose, lambose, galactose, and glucose. Non-reducing sugars include sucrose, trehalose, sorbose, melezitose, and raffinose. Mannitol, xylitol, erythritol, threitol, sorbitol, and glycerol are examples of sugar alcohols. With respect to sugar acids, these include L-gluconate and metal salts thereof. The polyol can also act as an isotonic agent. In certain embodiments of the present disclosure, one component of the formulation is sorbitol at a concentration of about 10 to about 70 mg / mL. In certain embodiments of the present disclosure, the concentration of sorbitol is from about 30 to about 50 mg / mL. In certain embodiments, the concentration of sucrose is from about 60 to about 100 mg / mL. In certain embodiments of the present disclosure, the concentration of sucrose is from about 70 to about 90 mg / mL.

The term "buffer" refers to a buffered solution that withstands changes in pH due to the action of its acid-base conjugated components. The buffer used in the present disclosure has a pH in the range of about 4.5 to about 7.5. Examples of buffers that will control the pH in this range include acetates (eg, sodium acetate), succinates (sodium succinate, etc.), gluconate, methionine, imidazole, histidine, glycine, arginine, Examples include citrates, phosphates, citrates and phosphates, Tris, and other organic acid buffers. In certain embodiments, the buffer used in the formulations of the present disclosure is histidine, glycine, arginine, acetate, citrate, and / or phosphate buffered saline (PBS).

A "reconstituted" formulation is one prepared by dissolving a lyophilized protein formulation in a diluent so that the protein is dispersed in the reconstituted formulation. The reconstituted formulation is suitable for administration (eg, parenteral administration) to a patient to be treated with the protein of interest (eg, LS-DVD-Ig).

The "diluents" as used herein are pharmaceutically acceptable (safe and non-toxic for administration to humans) and are useful in the preparation of liquid formulations such as those reconstituted after lyophilization. There is a diluent. Examples of diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffered solution (eg, phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solution. In certain alternative embodiments, the diluent can include an aqueous solution of salt and / or buffer.

A "therapeutically effective amount" or "effective amount" of a binding protein refers to an amount effective in the prevention or treatment of an antibody-effective disorder.

The term "disorder" refers to any condition that may benefit from treatment with the formulations of the present disclosure. This includes chronic and acute disorders or illnesses, including pathological conditions that make the subject susceptible to the disorder in question.

The term "treatment" refers to both therapeutic and prophylactic or proactive measures. Patients in need of treatment include those who already have a disability, as well as those whose disability should be prevented.

The terms "parenteral administration" and "parenteral administration" mean a mode of administration other than enteral and topical administration, usually by injection, without limitation, intravenously, intramuscularly, intraarterially, intramedullary. Includes intracavitary, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepithelial, intra-arterial, subcapsular, submucosal, intrathecal, and intrathoracic injections and injections. The terms "systemic", "systemic", "peripheral", and "peripheral" refer to a compound, drug, or other material that enters the patient's system, metabolizes and others. It means administration other than direct administration to the central nervous system, eg, subcutaneous administration, so that it is subject to a similar process.

The term "antibody" is broadly defined as an essential target binding of an immunoglobulin (Ig) molecule, or Ig molecule, generally consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains. Refers to any functional fragment, mutant, variant, or derivative thereof that retains its features.

In full-length antibodies, each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, namely CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), which are more conserved, called framework regions (FRs). The regions are interleaved. Each VH and VL consists of 3 CDRs and 4 FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can be of any type (eg IgG, IgE, IgM, IgD, IgA and IgY) and classes (eg IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclasses.

The term "Fc region" means the C-terminal region of an immunoglobulin heavy chain, which can be produced by papain digestion of intact antibodies. The Fc region may be a native sequence Fc region or a mutant sequence Fc region. The Fc region of an immunoglobulin generally comprises two constant domains, namely the CH2 and CH3 domains, optionally including the CH4 domain.

The term "antigen binding moiety" refers to one or more fragments of a binding protein that specifically bind to a target or antigen. Such embodiments may be unispecific, bispecific, bispecific, or multispecific (they may specifically bind to two or more different antigens).

A "functional antigen binding site" of a binding protein is a site capable of binding to a target antigen. The antigen-binding affinity of a functional antigen-binding site is not always as strong as the parent antibody from which the antigen-binding site is derived, but the ability to bind to an antigen assesses antibody binding to the antigen. Must be measurable using known methods for. Moreover, the antigen-binding affinities of each of the functional antigen-binding sites of a multivalent binding protein need not be quantitatively the same.

The term "linker" refers to a polypeptide containing two or more amino acid residues linked by a peptide bond, which is used to link one or more antigen binding moieties. Such linker polypeptides are well known in the art (eg, Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448, Poljak et al. (1994) Structure 2: 1121- See 1123).

"Immunoglobulin constant domain" refers to the heavy or light chain constant domain. Human heavy and light chain (eg, IgG) constant domain amino acid sequences are known in the art.

The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., with the exception of potential naturally occurring mutations in which individual antibodies containing that population may be present in small amounts. It is the same. Monoclonal antibodies are highly specific and are directed against a single antigen. Moreover, each monoclonal antibody is directed against a single epitope on the antigen, as opposed to polyclonal antibody preparations that typically contain different antibodies directed against different epitopes. The modifier "monoclonal" shall not be construed as requiring the production of an antibody by any particular method.

The term "human antibody" includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies contain amino acid residues that are not encoded by the human germline immunoglobulin sequence (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). Can include. However, the term "human antibody" is not intended to include an antibody in which a CDR sequence from the germline of another mammalian species, such as a mouse, is grafted onto a human framework sequence.

The term "chimeric antibody" refers to heavy and light chain variable region sequences from one species, such as antibodies with mouse heavy and light chain variable regions linked to human constant regions, as well as constants from another species. Means an antibody that comprises a region sequence.

The term "CDR graft antibody" is derived from one species, such as an antibody having human heavy and light chain variable regions in which one or more of the mouse CDRs (eg, CDR3) have been replaced with mouse CDR sequences. Means an antibody that comprises heavy and light chain variable region sequences, but in which one or more of the CDR regions of those VHs and / or VLs has been replaced with CDR sequences of another species.

The term "humanized antibody" includes heavy and light chain variable region sequences from non-human species (eg, mice), but at least a portion of the VH and / or VL sequence is more "human-like". That is, an antibody that has been altered to be more similar to a human germline variable sequence. One type of humanized antibody includes non-human CDR sequences and human framework sequences.

The term "CDR" means complementarity determining regions within an antibody variable sequence. There are three CDRs in each of the heavy and light chain variable regions, which are designated CDR1, CDR2, and CDR3 for each of the variable regions. As used herein, the term "CDR set" refers to a group of three CDRs that occurs in a single variable region that can bind to a target. The exact boundaries of these CDRs are defined differently by different systems.

The terms "Kabat numbering", "Kabat definition" and "Kabat labeling" are used interchangeably herein. These terms refer to a system of numbering amino acid residues that is more variable (ie, hypervariable) than amino acid residues in the heavy and light chain variable regions of the antibody or its antigen binding portion (Kabat et al. (1971) Ann. NY Acad. Sci. 190: 382-391 and Kabat et al. (1991) Chemicals of Proteins of Immunological Interest, Fifth Edition, U.S. Technology, Human Health 3242). With respect to the heavy chain variable region, the hypervariable region generally ranges from amino acids 31-35 for CDR1, amino acids 50-65 for CDR2, and amino acids 95-102 for CDR3. With respect to the light chain variable region, the hypervariable region generally ranges from amino acids 24-34 for CDR1, amino acids 50-56 for CDR2, and amino acids 89-97 for CDR3.

Conformatians and colleagues (Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917 and Chothia et al. (1989) Nature 342: 877-883) found that certain sub-parts within the Kabat CDR were amino acids. They have found that they have nearly identical peptide skeleton conformations, despite having greater diversity at the sequence level. These subparts were designated as L1, L2, and L3, or H1, H2, and H3 (where "L" and "H" refer to the light and heavy chain regions, respectively). These regions may be referred to as Chothia CDRs, which have boundaries that overlap with the Kabat CDRs. Other boundaries that define CDRs that overlap with Kabat CDRs are Padlan (1995) FASEB J. et al. 9: 133-139 and MacCallum (1996) J. Mol. Mol. Biol. 262 (5): 732-45. Yet other CDR boundary definitions may not strictly follow one of the above systems, but nevertheless overlap with Kabat CDRs, but they may be a particular residue or group of residues or even more. It can be shortened or prolonged in the light of predictive or experimental findings that the entire CDR does not significantly affect antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, but embodiments use CDRs defined by Kabat or Chothia.

The term "framework" or "framework sequence" refers to the rest of the sequence, which is the variable region minus the CDRs. Since the exact definition of a CDR sequence can be determined by different systems, the meaning of framework sequences is subject to different interpretations accordingly. The six CDRs (heavy chain CDRs-H1, -H2, and -H3, and light chain CDRs-L1, -L2, and -L3) also cross the framework regions on the light and heavy chains on each chain. Is divided into four subregions (FR1, FR2, FR3, and FR4), where CDR1 is between FR1 and FR2, CDR2 is between FR2 and FR3, and CDR3 is between FR3 and FR4. Positioned in between. The term "activity" includes activity such as binding specificity and binding affinity of DVD-Ig protein for two or more antigens.

The term "epitope" includes any polypeptide determinant capable of specifically binding to an immunoglobulin or T cell receptor. In certain embodiments, the epitope determinants include chemically active surface groups of molecules such as amino acids, sugar side chains, phosphoryls, or sulfonyls, and in certain embodiments, certain three-dimensional structural properties. , And / or may have specific charge characteristics. In certain embodiments, the epitope is a region of antigen that is bound by an antibody or multispecific binding protein.

The term "surface plasmon resonance" or "SPR" is used, for example, by detecting changes in protein concentration in a biosensor matrix using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). Refers to an optical phenomenon that enables real-time analysis of biomolecule-specific interactions. For further explanation, see Johnson et al. (1993) Ann. Biol. Clin. 51: 19-26, Johnson et al. (1991) Biotechniques 11: 620-627, Johnson et al. (1995) J.M. Mol. Recognit. 8: 125-131, and Johnsson et al. (1991) Anal. Biochem. 198: 268-277.

The term " _Kon " refers to the on-rate constant at which a binding protein associates with an antigen to form a binding protein / antigen complex, as is known in the art.

The term " _Koff " refers to the off-rate constant at which the binding protein dissociates from the binding protein / antigen complex, as is known in the art.

The term "K _d " refers to the dissociation constant of a particular antibody-antigen interaction, as is known in the art.

The terms "specific binding," "specifically binding," or "specifically binding," as used herein with reference to the interaction of a binding protein with a second species. Means that this interaction depends on the presence of a particular structure (eg, an antigenic determinant or epitope) on the species. For example, the DVD-Ig protein recognizes and binds to a unique protein structure rather than the protein in general. If the DVD-Ig protein is specific for epitope "A", then in the reactants containing labeled "A" and DVD-Ig protein, the molecule containing epitope A (or free unlabeled A) The presence will reduce the amount of labeled A bound to the DVD-Ig protein.

II. Double Variable Domain Immunoglobulin (DVD-Ig) Protein for Use in the Formulations of the Present Disclosure Although the present disclosure is specified as a DVD-Ig protein, particularly AS-DVD-Ig protein or LS-DVD-Ig protein. , Formulations, and their use (described in more detail below).

A. General DVD-Ig Protein Structure In certain embodiments, the DVD-Ig protein used in the formulations and methods of the present disclosure comprises a polypeptide chain, which polypeptide chain is VD1- (X1) n-VD2. Containing -C- (X2) n, in the formula, VD1 is the first variable domain, VD2 is the second variable domain, C is the constant domain, and X1 is the amino acid or polypeptide. , X2 represents the Fc region, and n is 0 or 1.

In certain embodiments, the DVD-Ig protein comprises two polypeptide chains, one of which comprises VD1- (X1) n-VD2-C- (X2) n in the formula. , VD1 is the first heavy chain variable domain, VD2 is the second heavy chain variable domain, C is the heavy chain constant domain, and X1 is the linker, provided that it is CH1. On condition that it is not, X2 is the Fc region, the second polypeptide comprises VD1- (X1) n-VD2-C- (X2) n, where VD1 is the first light chain. Variable domain, VD2 is the second light chain variable domain, C is the light chain constant domain, X1 is the linker, provided that it is not CH1 and X2 is the Fc region. On condition that it is not included, n is 0 or 1, and the two polypeptide chains of the binding protein form two functional antigen binding sites.

In certain embodiments, the DVD-Ig protein contains four polypeptide chains, of which two polypeptide chains contain VD1- (X1) n-VD2-C- (X2) n and formulate. Among them, VD1 is the first heavy chain variable domain, VD2 is the second heavy chain variable domain, C is the heavy chain constant domain, and X1 is the linker, but it is Given that it is not CH1, X2 is the Fc region, the two polypeptide chains contain VD1- (X1) n-VD2-C- (X2) n, where VD1 is the first light. Chain variable domain, VD2 is the second light chain variable domain, C is the light chain constant domain, X1 is the linker, provided that it is not CH1 and X2 is the Fc region. N is 0 or 1, and the four polypeptide chains of the binding protein form four functional antigen-binding sites, provided that they do not contain.

In certain embodiments, the AS-DVD-Ig protein or LS-DVD-Ig protein comprises a polypeptide chain, which polypeptide chain comprises VD1- (X1) n-VD2-C- (X2) n. In the formula, VD1 is the first heavy chain variable domain, VD2 is the second heavy chain variable domain, C is the heavy chain constant domain, and X1 is the linker, but , X2 is the Fc region and n is 0 or 1, provided it is not CH1. In certain embodiments, the AS-DVD-Ig protein or LS-DVD-Ig protein comprises a polypeptide chain, which polypeptide chain comprises VD1- (X1) n-VD2-C- (X2) n. In the formula, VD1 is the first light chain variable domain, VD2 is the second light chain variable domain, C is the light chain constant domain, and X1 is the linker, but , X2 does not contain the Fc region, and n is 0 or 1, provided that it is neither CH1 nor CL. In a further embodiment, (X1) n on the heavy and / or light chain is (X1) 0 and / or (X2) n on the heavy and / or light chain is (X2) 0. is there.

In certain embodiments, the AS-DVD-Ig protein or LS-DVD-Ig protein comprises first and second polypeptide chains, of which the first polypeptide chain is the first VD1-(. X1) contains n-VD2-C- (X2) n, where VD1 is the first heavy chain variable domain, VD2 is the second heavy chain variable domain, and C is the heavy chain constant. The domain, X1 is the first linker, provided that it is not CH2, X2 is the Fc region, n is 0 or 1, and the second polypeptide chain is. , 2nd VD1- (X1) n-VD2-C- (X2) n, in the formula VD1 is the first light chain variable domain and VD2 is the second light chain variable domain. , C is the light chain constant domain, X1 is the second linker, provided that it is neither CH1 nor CL, X2 does not contain the Fc region and n is 0 or It is 1.

In certain embodiments, VD1 of the first polypeptide chain and VD1 of the second polypeptide chain are derived from different first and second parent antibodies, or their binding moieties, respectively. In certain embodiments, the VD2 of the first polypeptide chain and the VD2 of the second polypeptide chain are derived from different first and second parent antibodies, or their binding moieties, respectively. In certain embodiments, the first and second parent antibodies bind to different epitopes on the same target or different targets. In certain embodiments, the first parent antibody or binding moiety thereof binds to the first target with a different potency than the potency of the second parent antibody or binding moiety thereof binding to the second target. In certain embodiments, the first parent antibody or binding moiety thereof binds to the first target with an affinity different from the affinity by which the second parent antibody or binding moiety thereof binds to the second target. To do.

In certain embodiments, the AS-DVD-Ig protein or LS-DVD-Ig protein comprises two first polypeptide chains and two second polypeptide chains.

In certain embodiments, the AS-DVD-Ig protein or LS-DVD-Ig protein comprises first and second polypeptide chains, each independently of VD1- (X1) n-VD2-C-. In the formula, VD1 is the first variable domain, VD2 is the second variable domain, C is the constant domain, and X1 is the linker, except that (X2) n is included. Given that it is not CH1, X2 is the Fc region, n is 0 or 1, and the VD1 domain on this first and second polypeptide chain is the first functional target binding. The site is formed and the VD2 domain on the first and second polypeptide chains forms a second functional target binding site. In a further embodiment, the first polypeptide chain comprises a first VD1- (X1) n-VD2-C- (X2) n, where VD1 is the first heavy chain variable domain in the formula. VD2 is the second heavy chain variable domain, C is the heavy chain constant domain, X1 is the linker, provided that it is not CH1 and X2 is the Fc region. n is 0 or 1, the second polypeptide chain comprises a second VD1- (X1) n-VD2-C- (X2) n, where VD1 is the first light chain variable in the formula. A domain, VD2 is a second light chain variable domain, C is a light chain constant domain, X1 is a linker, provided that it is not CH1 and X2 is an Fc. Not including a region, n is 0 or 1, and the VD1 domain on the first and second polypeptide chains forms a first functional target binding site, the first and second poly. The VD2 domain on the peptide chain forms a second functional target binding site.

In certain embodiments, the AS-DVD-Ig protein or LS-DVD-Ig protein comprises first and second polypeptide chains, each independently of VD1- (X1) n-VD2-C-. In the formula, VD1 is the first variable domain, VD2 is the second variable domain, C is the constant domain, and X1 is the linker, except that (X2) n is included. Given that it is not CH1, X2 is the Fc region, n is 0 or 1, and the VD1 domain on this first and second polypeptide chain is the first functional target binding. The site is formed and the VD2 domain on the first and second polypeptide chains forms a second functional target binding site. In a further embodiment, the first polypeptide chain comprises a first VD1- (X1) n-VD2-C- (X2) n, where VD1 is the first heavy chain variable domain in the formula. VD2 is the second heavy chain variable domain, C is the heavy chain constant domain, X1 is the linker, provided that it is not CH1 and X2 is the Fc region. n is 0 or 1, the second polypeptide chain comprises a second VD1- (X1) n-VD2-C- (X2) n, where VD1 is the first light chain variable in the formula. A domain, VD2 is a second light chain variable domain, C is a light chain constant domain, X1 is a linker, provided that it is not CH1 and X2 is an Fc. Not including a region, n is 0 or 1, and the VD1 domain on the first and second polypeptide chains forms a first functional target binding site, the first and second poly. The VD2 domain on the peptide chain forms a second functional target binding site.

Examples of DVD-Ig proteins are described in US Pat. No. 7,612,181, which is incorporated herein by reference.

Examples of DVD-Ig proteins that can be used in the methods and compositions of the present disclosure are listed below, including Tables 58 and 65. Further examples of DVD-Ig proteins that can be used in the methods and compositions of the present disclosure are set forth in SEQ ID NOs: 28-75.

B. Production of DVD-Ig Protein The variable domain of the DVD-Ig protein can be obtained from the parent antibody, including polyclonal and monoclonal antibodies capable of binding to the target of interest. These antibodies may be naturally occurring or may be produced by recombinant techniques. Examples of antibodies that can be used to make the DVD-Ig protein include chimeric antibodies, human antibodies, and humanized antibodies. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art, such as the use of hybridoma, recombination, and phage display techniques, or any combination thereof. Monoclonal antibodies also among human immunoglobulin loci, such as the engineered mouse strain XENOMOUSE (TM) transgenic mouse, which contains a large fragment of the human immunoglobulin locus and is deficient in mouse antibody production. It may be produced by immunizing a non-human animal, including some or all, with the antigen of interest. Methods for producing DVD-Ig proteins are described in US Pat. No. 7,612,181, and the teachings of this patent are incorporated herein by reference. The DVD-Ig protein used in the compositions and methods of the present disclosure is capable of binding to a particular target and may be made from antibodies well known in the art. These include anti-TNF antibody (US Pat. No. 6,258,562), anti-IL-12 and / or anti-IL-12p40 antibody (US Pat. No. 6,914,128); anti-IL-18 antibody (US Pat. No. 6,914,128). Publication No. 20050147610), as well as anti-C5, anti-CBL, anti-CD147, anti-gp120, anti-VLA4, anti-CD11a, anti-CD18, anti-VEGF, anti-CD40L, anti-Id, anti-ICAM-1, anti-CXCL13, anti-CD2, anti-EGFR , Anti-TGF-β2, anti-E-selectin, anti-Fact VII, anti-Her2 / neu, anti-F gp, anti-CD11 / 18, anti-CD14, anti-ICAM-3, anti-CD80, anti-CD4, anti-CD3, anti-CD23, anti β2-Integrin, anti-α4β7, anti-CD52, anti-HLA DR, anti-CD22, anti-CD20, anti-MIF, anti-CD64 (FcR), anti-TCR αβ, anti-CD2, anti-Hep B, anti-CA 125, anti-EpCAM, anti-gp120, Anti-CMV, anti-gpIIbIIIa, anti-IgE, anti-CD25, anti-CD33, anti-HLA, anti-VNR integrin, anti-IL-1α, anti-IL-1β, anti-IL-1 receptor, anti-IL-2 receptor, anti-IL-4 , Anti-IL4 receptor, anti-IL5, anti-IL-5 receptor, anti-IL-6, anti-IL-8, anti-IL-9, anti-IL-13, anti-IL-13 receptor, anti-IL-17, and anti. IL-23 antibodies are included, but not limited to (Presta (2005) J. Allergy Clin. Immunol. 116: 731-6 and Clark “Antibodies for Therapeutic Applications,” “Depertment for Technology Applications,” Please refer to (Published online on M. Clark's homepage on the website of Department of Pathology, Cambridge University).

The parental monoclonal antibody may also be selected from a variety of therapeutic antibodies that have been approved for use in clinical trials or are under development for clinical use. Such therapeutic antibodies include, but are not limited to: rituzimab (RITUXAN (TM) Biogen Idec, Genentech / Roche) (see, eg, US Pat. No. 5,736,137). That is, a chimeric anti-CD20 antibody approved for the treatment of non-Hodgkin lymphoma; ofutumumab (HUMAX-CD20 (TM) Genmab, GlaxoSmisKlein) (described in US Pat. No. 5,500,362), ie, fludarabin and Anti-CD20 antibody approved for the treatment of chronic lymphocytic leukemia refractory to alemtuzumab; AME-133v (Mentrik Biotech) or anti-CD20 antibody; pertuzumab (hA20) (Immunomedics) or anti-CD20 antibody; HumaLYM (Intracel) PRO70769 (Genentech / Roche) (PCT / US Patent Publication No. 2003/04426) or anti-CD20 antibody; trastuzumab (HERCEPTIN (TM) Genentech / Roche) (described in US Pat. No. 5,677,171) or Humanized anti-Her2 / neu antibody approved for the treatment of breast cancer; pertuzumab (rhuMab-2C4, OMNITALG (TM) Genentech / Roche) (described in US Pat. No. 4,753,894); cetuximab (ERBITUX) (TM) Imclone) (described in US Pat. No. 4,943,533, PCT WO 96/40210), a chimeric anti-EGFR antibody approved for the treatment of colorectal and head and neck cancers; Panitumumab (ABX-EGF VECTIBIX (R) Amen) (described in US Pat. No. 6,235,883), an anti-EGFR antibody approved for the treatment of colorectal cancer; saltumumab (HUMAX-EGFR (TM)) Genmab) (described in US Patent Application No. 10 / 172,317) or anti-EGFR antibody; EMD55900 (Mab 425 Markk) or anti-EGFR antibody; EMD62000 and EMD72000 (Mab 425 Mark) or anti-EGFR antibody (US Patent No. 1). 5,558,864, Murthy et al. (1987) Arch. Biochem. Biophyss. 252 (2) : 549-60, Rodeck et al. (1987) J.M. Cell. Biochem. 35 (4): 315-20, Kettreborogue et al. (1991) Protein Eng. 4 (7): 773-83; ICR62 (Institute of Cancer Research) or anti-EGFR antibody (PCT Publication No. WO 95/20054, Modjtahedi et al. (1993) J. Cell. Biophyss. 22 (1993). 1-3): 129-46, Modjtahedi et al. (1993) Br. J. Cancer 67 (2): 247-53, Modjtahedi et al. (1996) Br. J. Cancer 73 (2): 228-35. , Modjtahedi et al. (2003) Int. J. Cancer 105 (2): 273-80); Nimotuzumab (TheraCIM hR3, THERALOC (R) YM Biosciences, Oncoscience AG) (US Pat. No. 5,89). 996, US Pat. No. 6,506,883, Mateo et al. (1997) Immunotechnol. 3 (1): 71-81) ie anti-EGFR antibody; ABT-806 (Ludwig Institute for Cancer Research, Memorial Sloan-Kettering (Jungblue et al. (2003) Proc. Natl. Acad. Sci. USA 100 (2): 639-44) or anti-EGFR antibody; KSB-102 (KS Biomedix); MR1-1 (IVAX, IVAX, National Cancer Institute (PCT Publication No. WO 0162931A2) or anti-EGFRvIII antibody; SC100 (Cancell) (PCT Publication No. WO 01/88138) or anti-EGFR antibody; Alemtuzumab (CAMPATH (TM) Genzyme / Sanofi) or B-cell Anti-CD52 antibody approved for the treatment of lymphocytic leukemia; Muromonab-CD3 (Orthoclone OKT3 (TM) Johnson and Johnson), an anti-CD3 antibody approved for the treatment of organ transplant rejection; Ibritumo mabutiuxetan (ZEVALIN (TM) Spectram Pharmaceuticals), an anti-CD20 antibody approved for the treatment of non-Hodgkin lymphoma; gemtuzumab ozogamicin (hP67.6 MYLOTARG (TM) P) phizer) or anti-CD33 antibody complexed with calikeamycin; alefacept (AMEVIVE (TM) Astellas Pharma) or anti-CD2 LFA-3 Fc fusion; absikimab (REOPRO (TM) Centocor Ortho Biotech heart, Lil) Chimeric human-mouse glycoprotein IIb / IIIa receptor and anti-bitronectin α _v β ₃ receptor antibody approved as an adjunct to percutaneous coronary angioplasty to prevent blood (cardiac ishemia); basiliximab (SIMULECT (TM)) Novartis), an anti-CF25 antibody approved to treat organ transplant rejection; paribismab (SYNAGIS (TM) Medium), an antibody to the A antigen site of the RSV F protein approved to treat RSV infection; infliximab (REMICADE (TM) Janssen Biotech), an anti-TNFαα antibody approved for the treatment of Crohn's disease, ulcerative colitis, arthritis, tonic spondylitis, psoriatic arthritis, and psoriasis vulgaris; adalimumab (HUMIRA (TM)) AbbVie) i.e. Anti-TNFα antibody approved to treat rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, tonic spondylitis, Crohn's disease, ulcerative colitis, psoriasis vulgaris; CDP571 (HUMICADE (TM)) Celltech, Biogen IDEC) Anti-TNFα antibody; Eternelcept (ENBREL (TM) Amen, Pphizer) or rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, tonic spondylitis, psoriasis vulgaris TNFαFc fusion antibody; Celtrizumab Pegor (CIMZIA) UCB Pharma) or anti-TNFα antibody approved for the treatment of rheumatoid arthritis and Crohn's disease; STELARA Janssen Biotech or psoriasis vulgaris Human anti-p40 subsystem of IL-12 and IL-23 antibodies; galilimomab (ABX-CBL Abgenix) or mouse anti-CD147 antibody; ABX-IL8 (Abgenix) or anti-IL8 antibody; ABX-MA1 (Abgenix) or Anti-MUC18 antibody; pemtu momab) (Theergyn, R1549, 90Y-muHMFG1 Antisoma) or mouse anti-MUC1-itrium 90 antibody complex; Therex (R1550 Antisoma) or anti-MUC1 antibody; AngioMab (muBC-1, AsaTima; (AS1407 Antisoma); Natarizumab (TYSABRI (R) Biogen Idec, Elan), an anti-α4 integrin antibody approved for the treatment of multiple sclerosis and Crohn's disease; VLA-1 (Santarus), a humanized anti-VLA-1. Antibodies; LTBR mAb (Biogen Idec) or anti-phosphotoxin β-receptor antibody; Lerdelimumab (CAT-152 Cambridge Antibody Technology / Abbott) or anti-TGF-β2 antibody; Briaquinumab (Ab-i-Mab) -192 Cambridge antibody Technology, Genzyme) i.e. anti-TGFβ1 antibodies; Beruchirimumabu (CAT-213, iCO-008 Cambridge antibody Technology, iCo Therapeutics, Immune Pharmaceuticals) i.e. anti-eotaxin 1 antibody; belimumab (BENLYSTA (R) GlaxoSmithKline) i.e. systemic lupus erythematosus Anti-B lymphocyte stimulating factor protein antibody approved to treat the disease; maptumumab (HGS-ETR1 Cambridge Antibody Technology, human Genome Sciences) or anti-TRAIL-R1 antibody (anti-TRAIL-R1 antibody) That is, an anti-VEGF antibody approved for treating metastatic colorectal cancer, non-squamous non-small cell lung cancer, glioblastoma, and metastatic renal cell carcinoma; anti-HER3 / EGFR antibody (Genentech / Roche); anti-tissue factor Antibodies (Genentech / Roche); Omarizumab (XOLAIR (TM) Genentech / Roche, Novalis), an anti-IgE antibody approved for the treatment of severe allergic asthma; ALIzumab (RAPTIVA (TM) Genentech / Roche, Merck Serono) or anti-CD11a antibody; MLN-02 (Millenium, Genentech / Roche) or anti-α4β7 integran antibody; zanolimumab (HUMAX CD4 (TM) Anti-CD4 -IL15 (TM) (AMG-714 Genmab, Agen) or anti-IL15 antibody; HuMax-IL8 (HUMAX-Inflam (TM), MDX-018 Genmab, Cormorant Pharmaceuticals) or anti-IL8 antibody; HUMAX (TM) -Can Genmab, Medarex, Oxford GlycoSciences) or anti-heparanase I antibody; HUMAX (TM) -Lymphoma (Genmab) or anti-IL8 antibody; HUMAX (TM) -TAC (Genmab) or anti-IL-2Rα, CD25 antibody; )-CD38, Genmab, Janssen Biotech) or anti-CD38 antibody; tralizumab (IDEC-131 Biogen Idec) or anti-CD40L antibody; clenolimimab (IDEC-151 Biogen Idec) or anti-CD4 antibody (IDEC-131 Biogen Idec). 114 Biogen Idec or anti-CD80 antibody; lumilixmab (IDEC-152 Biogen Idec) or anti-CD23; anti-macrophage migrating factor (MIF) antibody (Biogen Idec, Taisho Pharmaceutic) anti-Maizo Pharmaceutic Antibodies; IMC-1C11 (Imclone) or chimeric anti-VEGFR2 antibody; DC101 (Imclone) or mouse anti-VEGFR2 antibody ;; anti-VE cadoherin antibody (Imclone); Eplatsumab (LYMPHOCIDE (TM) Immunomedics) or anti-CD22 antibody; Takatsuzumab tetraxetaneittrium ( ⁹⁰ Y) (AFP-Cide (R) Im) Munomedics, an anti-α fetal protein antibody; MyelomaCide (R) Immunomedics, or anti-CF74 antibody; LeukoCide (R) (Immunomedics); ProstaCide (R) (Immunomedics); ImmunoMedics; -Myers Squibb) or anti-CTLA4 antibody approved for the treatment of melanoma; iratumumab (MDX-060 Medarex) or anti-CD30 antibody; MDX-070 (Medarex) or antiprostatic specific membrane antigen; OSIDEM (TM) ( IDM-1 Medarex, Immuno-Designed Moleculars) or anti-Her2 antibody; HUMAX (TM) -CD4, i.e. anti-CD4 antibody developed by Medarex and Genmab; HuMax-IL15, ie anti-IL15 developed by Medarex and Genmab. Antibodies; SIMPONI (TM) Janssen Biotech or anti-TNFα antibody approved for the treatment of rheumatoid arthritis, psoriatic arthritis, tonic spondylitis; Ustequinumab (STELARA (R), CNTO 1275 Janssen Biotech) or vulgaris Anti-IL-12 antibody approved to treat psoriasis; MOR101 and MOR102 (MorphoSystems) or anti-cell adhesion molecule-1 (ICAM-1) (CD54) antibody; MOR201 (MorphoSys) or anti-fibroblast growth factor acceptance Body 3 antibody; vivilizumab (NUVION (TM) PDL BioPharma) or anti-CD3 antibody; font lizamab (HUZAF (TM) PDL BioPharma) or anti-INFγ antibody; borosiximab (M200 PDL BioPharma or anti-CD3 antibody) (R) IL-12 (PDL BioPharma) or anti-IL-12; ING-1 (Xoma) or anti-Ep-CAM antibody; omalizumab (XOLAIR (TM) Genentech / Roche, Novartis) or allergic asthma Approved anti-IgE antibody; MLN01 (Xoma) or anti-β integrin antibody; and Tosirizuma Bu (ACTEMRA (TM) Genentech / Roche), an anti-IL6 antibody approved for the treatment of rheumatoid arthritis and systemic juvenile idiopathic arthritis.

C. Construction of DVD-Ig Protein DVD-Ig protein is formed by combining two heavy chain DVD polypeptides and two light chain DVD polypeptides. The bivariable domain immunoglobulin (DVD-Ig) heavy chain comprises two heavy chain variable domains (VHs) linked directly or in series by a linker, followed by constant domains CH1 and Fc regions. A double variable domain immunoglobulin (DVD-Ig) light chain consists of two light chain variable domains (VL) from two parent mAbs linked in series, either directly or via a linker, followed by a light chain constant domain (CL). ) (See Figure 1A of US Pat. No. 7,612,181, which is incorporated herein by reference). Methods for making DVD-Ig proteins are also described in US Pat. No. 7,612,181 and are incorporated herein by reference.

The variable domain of the DVD-Ig protein can be obtained using recombinant DNA techniques from the parent antibody produced by any one of the methods described above. In certain embodiments, the variable domain is a CDR graft or a humanized variable heavy or light chain domain. In certain embodiments, the variable domain is a human heavy or light chain variable domain.

The linker sequence may be a single amino acid sequence or a polypeptide sequence. Examples of linker sequences that can be used to link variable domains are AKTTPKLEEGFSEAR (SEQ ID NO: 1), AKTTPKLEEGEFSEARV (SEQ ID NO: 2), AKTTPKLGG (SEQ ID NO: 3), SAKTTPKLGG (SEQ ID NO: 4), SAKTTP (SEQ ID NO: 5). ), RADAAP (SEQ ID NO: 6), RADAAPTVS (SEQ ID NO: 7), RADAAAAGGPGS (SEQ ID NO: 8), RADAAAAA (G _4S ). ₄ (SEQ ID NO: 9), SAKTTPCLEEGEFSEARV (SEQ ID NO: 10), ADAAP (SEQ ID NO: 11), ADAAPTVSIFPP (SEQ ID NO: 12), TVAAP (SEQ ID NO: 13), TVAAPSVFIFPP (SEQ ID NO: 14), QPKAAP (SEQ ID NO: 15), QPKAAPSVTLFPP (SEQ ID NO: 16), AKTTPP (SEQ ID NO: 17), AKTTPPSPVTPLAP (SEQ ID NO: 18), AKTTAP (SEQ ID NO: 19), AKTTAPPSVYPLAP (SEQ ID NO: 20), ASTKGP (SEQ ID NO: 21), ASTKGPSVFPLAP (SEQ ID NO: 22), GGGGGSGGGSGGG SEQ ID NO: 23), GENKVEYAPALMALS (SEQ ID NO: 24), GPAKELTPLKEAKVS (SEQ ID NO: 25), GHEAAAVMQVQYPAS (SEQ ID NO: 26), and GGGGSGGGGGS (SEQ ID NO: 27), but not limited to these. Other examples of linkers are described in US Patent Publication No. 2010226923 and are incorporated herein by reference. The selection of the linker sequence may be determined based on the crystal structure analysis of some antibody Fab molecules. There is a natural mobile chain between the variable domain and the CH1 / CL constant domain in the Fab or antibody molecular structure. This natural chain consists of approximately 10-12 amino acid residues provided by 4-6 residues from the C-terminus of the V domain and 4-6 residues from the N-terminus of the CL or CH1 domain. including. The DVD-Ig proteins of the present disclosure use 5 to 6 amino acid residues or 11 to 12 amino acid residues at the N-terminus of CL or CH1 as linkers in the light and heavy chains of the DVD-Ig protein, respectively. Was generated. The N-terminal residues of the CL or CH1 domains, especially the first 5-6 amino acid residues, adopt a loop conformation without the use of strong secondary structure and thus a mobile linker between the two variable domains. Can act as. The N-terminal residues of the CL or CH1 domain are a natural extension of the variable domain because they are part of the Ig sequence, and thus the immunogenicity that can arise from its linker or junction is minimal. Be transformed. Further examples of linkers are shown in Table 65 (see Underlined Amino Acids).

Other linker sequences are not all residues of the CL / CH1 domain, but may contain sequences of any length in the CL or CH1 domain, eg, the first 5-12 amino acid residues of the CL or CH1 domain. That is, the light chain linker can be from Cκ or Cλ, and the heavy chain linker is derived from any isotype of CH1, including Cγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα2, Cδ, Cε, and Cμ. Can be done. Linker sequences are also Ig-like proteins (eg, TCR, FcR, KIR), G / S-based sequences (eg, G4S repeats); other proteins such as hinge region-derived sequences and other natural sequences derived from the protein. It may be derived from.

In certain embodiments, the constant domain is linked into two linked variable domains using recombinant DNA techniques. For example, a sequence containing a linked heavy chain variable domain is linked to a heavy chain constant domain, and a sequence containing a linked light chain variable domain is linked to a light chain constant domain. In certain embodiments, the constant domains are the human heavy chain constant domain and the human light chain constant domain, respectively. In certain embodiments, the DVD-Ig heavy chain is further linked to the Fc region. The Fc region may include a native Fc region sequence or a mutant Fc region sequence. In certain embodiments, the Fc region is a human Fc region. For example, the Fc region comprises an Fc region from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD.

In certain embodiments, the DVD-Ig protein used in the methods and compositions of the present disclosure is a bispecific tetravalent binding protein. In certain embodiments, the DVD-Ig protein used in the methods and compositions of the present disclosure binds to CD20 and CD80, and in certain embodiments, the DVD used in the methods and compositions of the present disclosure. -Ig protein binds to VEGF and HER2. In certain embodiments, the DVD-Ig protein used in the methods and compositions of the present disclosure binds to TNF and RANKL. In certain embodiments, the DVD-Ig protein used in the methods and compositions of the present disclosure binds to TNF and DKK. In certain embodiments, the DVD-Ig protein used in the methods and compositions of the present disclosure binds to CD20 and RANKL. In certain embodiments, the DVD-Ig protein used in the methods and compositions of the present disclosure binds to PLL4 and PLGF. In certain embodiments, the DVD-Ig protein used in the methods and compositions of the present disclosure binds to PLL4 and VEGF. In certain embodiments, the DVD-Ig protein used in the methods and compositions of the present disclosure binds to TNF and SOST. In certain embodiments, the DVD-Ig protein used in the methods and compositions of the present disclosure binds IL-9 and IgE. In certain embodiments, the DVD-Ig protein used in the methods and compositions of the present disclosure binds IL-12 and IL-18. Examples of IL-12 and IL-18 DVD-Ig proteins are described in US Pat. No. 7,612,181. In certain embodiments, the DVD-Ig protein used in the methods and compositions of the present disclosure binds to TNF and IL-17. In certain embodiments, the DVD-Ig protein used in the methods and compositions of the present disclosure binds to TNF and PGE2. An example of the PGE2 DVD-Ig protein is provided in US Patent Publication No. 20100074900. In certain embodiments, the DVD-Ig protein used in the methods and compositions of the present disclosure binds IL-1α and IL-1β. Examples of IL-1α and IL-1β DVD-Ig proteins are described in US Pat. No. 7,612,181. In certain embodiments, the DVD-Ig protein used in the methods and compositions of the present disclosure binds IL-4 and IL-1. Examples of IL-4 and IL-13 DVD-Ig proteins are described in US Publication No. 2010226923. The amino acid and nucleic acid sequences of the DVD-Ig proteins described in the above patents and patent applications are incorporated herein by reference. The amino acid sequences of the DVD-Ig proteins that can be used in the methods and compositions of the present disclosure are set forth in SEQ ID NOs: 28-75.

In certain embodiments, the DVD-Ig protein used in the methods and compositions of the present disclosure specifically binds to CD20 / CD80 to the heavy and light chain CDRs set forth in SEQ ID NOs: 30 and 31. Contains the corresponding amino acid sequence. In certain embodiments, the anti-CD20 / CD80 DVD-Ig protein comprises the amino acid sequences corresponding to the heavy and light chain variable regions set forth in SEQ ID NOs: 30 and 31. In certain embodiments, the anti-CD20 / CD80 DVD-Ig protein comprises the amino acid sequences corresponding to the heavy and light chains set forth in SEQ ID NOs: 30 and 31.

In certain embodiments, the DVD-Ig protein used in the methods and compositions of the present disclosure specifically binds to CD80 / CD20 into the heavy and light chain CDRs set forth in SEQ ID NOs: 32 and 33. Contains the corresponding amino acid sequence. In certain embodiments, the anti-CD80 / CD20 DVD-Ig protein comprises the amino acid sequences corresponding to the heavy and light chain variable regions set forth in SEQ ID NOs: 32 and 33. In certain embodiments, the anti-CD80 / CD20 DVD-Ig protein comprises the amino acid sequences corresponding to the heavy and light chains set forth in SEQ ID NOs: 32 and 33.

In certain embodiments, the DVD-Ig protein used in the methods and compositions of the present disclosure specifically binds to VEGF / HER2 to the heavy and light chain CDRs set forth in SEQ ID NOs: 34 and 35. Contains the corresponding amino acid sequence. In certain embodiments, the anti-VEGF / HER2-Ig protein comprises the amino acid sequences corresponding to the heavy and light chain variable regions set forth in SEQ ID NOs: 34 and 35. In certain embodiments, the anti-VEGF / HER2 DVD-Ig protein comprises the amino acid sequences corresponding to the heavy and light chains set forth in SEQ ID NOs: 34 and 35.

In certain embodiments, the DVD-Ig protein used in the methods and compositions of the present disclosure specifically binds to HER2 / VEGF into the heavy and light chain CDRs set forth in SEQ ID NOs: 36 and 37. Contains the corresponding amino acid sequence. In certain embodiments, the anti-HER2 / VEGF-Ig protein comprises the amino acid sequences corresponding to the heavy and light chain variable regions set forth in SEQ ID NOs: 36 and 37. In certain embodiments, the anti-HER2 / VEGF DVD-Ig protein comprises the amino acid sequences corresponding to the heavy and light chains set forth in SEQ ID NOs: 36 and 37.

In certain embodiments, the DVD-Ig protein used in the methods and compositions of the present disclosure specifically binds to TNF / RANKL to the heavy and light chain CDRs set forth in SEQ ID NOs: 38 and 39. Contains the corresponding amino acid sequence. In certain embodiments, the anti-TNF / RANKL-Ig protein comprises the amino acid sequences corresponding to the heavy and light chain variable regions set forth in SEQ ID NOs: 38 and 39. In certain embodiments, the anti-TNF / RANKL DVD-Ig protein comprises the amino acid sequences corresponding to the heavy and light chains set forth in SEQ ID NOs: 38 and 39.

In certain embodiments, the DVD-Ig protein used in the methods and compositions of the present disclosure specifically binds to RANKL / TNF into the heavy and light chain CDRs set forth in SEQ ID NOs: 40 and 41. Contains the corresponding amino acid sequence. In certain embodiments, the anti-RANKL / TNF-Ig protein comprises the amino acid sequences corresponding to the heavy and light chain variable regions set forth in SEQ ID NOs: 40 and 41. In certain embodiments, the anti-RANKL / TNF DVD-Ig protein comprises the amino acid sequences corresponding to the heavy and light chains set forth in SEQ ID NOs: 40 and 41.

In certain embodiments, the DVD-Ig protein used in the methods and compositions of the present disclosure specifically binds to TNF / DKK into the heavy and light chain CDRs set forth in SEQ ID NOs: 42 and 43. Contains the corresponding amino acid sequence. In certain embodiments, the anti-TNF / DKK-Ig protein comprises the amino acid sequences corresponding to the heavy and light chain variable regions set forth in SEQ ID NOs: 42 and 43. In certain embodiments, the anti-TNF / DKK-Ig protein comprises the amino acid sequences corresponding to the heavy and light chains set forth in SEQ ID NOs: 42 and 43.

In certain embodiments, the DVD-Ig protein used in the methods and compositions of the present disclosure specifically binds to DKK / TNF to the heavy and light chain CDRs set forth in SEQ ID NOs: 44 and 45. Contains the corresponding amino acid sequence. In certain embodiments, the anti-DKK / TNF-Ig protein comprises the amino acid sequences corresponding to the heavy and light chain variable regions set forth in SEQ ID NOs: 44 and 45. In certain embodiments, the anti-DKK / TNF-Ig protein comprises the amino acid sequences corresponding to the heavy and light chains set forth in SEQ ID NOs: 44 and 45.

In certain embodiments, the DVD-Ig protein used in the methods and compositions of the present disclosure specifically binds to CD20 / RANKL to the heavy and light chain CDRs set forth in SEQ ID NOs: 46 and 47. Contains the corresponding amino acid sequence. In certain embodiments, the anti-CD20 / RANKL-Ig protein comprises the amino acid sequences corresponding to the heavy and light chain variable regions set forth in SEQ ID NOs: 46 and 47. In certain embodiments, the anti-CD20 / RANKL-Ig protein comprises the amino acid sequences corresponding to the heavy and light chains set forth in SEQ ID NOs: 46 and 47.

In certain embodiments, the DVD-Ig protein used in the methods and compositions of the present disclosure specifically binds to RANKL / CD20 into the heavy and light chain CDRs set forth in SEQ ID NOs: 48 and 49. Contains the corresponding amino acid sequence. In certain embodiments, the anti-RANKL / CD20-Ig protein comprises the amino acid sequences corresponding to the heavy and light chain variable regions set forth in SEQ ID NOs: 48 and 49. In certain embodiments, the anti-RANKL / CD20-Ig protein comprises the amino acid sequences corresponding to the heavy and light chains set forth in SEQ ID NOs: 48 and 49.

In certain embodiments, the DVD-Ig protein used in the methods and compositions of the present disclosure specifically binds to PLL4 / PLGF into the heavy and light chain CDRs set forth in SEQ ID NOs: 50 and 51. Contains the corresponding amino acid sequence. In certain embodiments, the anti-DLL4 / PLGF-Ig protein comprises the amino acid sequences corresponding to the heavy and light chain variable regions set forth in SEQ ID NOs: 50 and 51. In certain embodiments, the anti-DLL4 / PLGF-Ig protein comprises the amino acid sequences corresponding to the heavy and light chains set forth in SEQ ID NOs: 50 and 51.

In certain embodiments, the DVD-Ig protein used in the methods and compositions of the present disclosure specifically binds PLGF / PLL4 to the heavy and light chain CDRs set forth in SEQ ID NOs: 52 and 53. Contains the corresponding amino acid sequence. In certain embodiments, the anti-PLGF / PLL4-Ig protein comprises the amino acid sequences corresponding to the heavy and light chain variable regions set forth in SEQ ID NOs: 52 and 53. In certain embodiments, the anti-PLGF / PLL4-Ig protein comprises the amino acid sequences corresponding to the heavy and light chains set forth in SEQ ID NOs: 52 and 53.

In certain embodiments, the DVD-Ig proteins used in the methods and compositions of the present disclosure specifically bind to TNF / SOST (S2) and are heavy and light chains set forth in SEQ ID NOs: 54 and 55. Contains the amino acid sequence corresponding to the chain CDR. In certain embodiments, the anti-TNF / SOST (S2) -Ig protein comprises the amino acid sequences corresponding to the heavy and light chain variable regions set forth in SEQ ID NOs: 54 and 55. In certain embodiments, the anti-TNF / SOST (S2) -Ig protein comprises the amino acid sequences corresponding to the heavy and light chains set forth in SEQ ID NOs: 54 and 55.

In certain embodiments, the DVD-Ig proteins used in the methods and compositions of the present disclosure specifically bind SOST (S2) / TNF and are heavy and light chains set forth in SEQ ID NOs: 56 and 57. Contains the amino acid sequence corresponding to the chain CDR. In certain embodiments, the anti-SOST (S2) / TNF-Ig protein comprises the amino acid sequences corresponding to the heavy and light chain variable regions set forth in SEQ ID NOs: 56 and 57. In certain embodiments, the anti-SOST (S2) / TNF-Ig protein comprises the amino acid sequences corresponding to the heavy and light chains set forth in SEQ ID NOs: 56 and 57.

In certain embodiments, the DVD-Ig protein used in the methods and compositions of the present disclosure specifically binds IL-9 (S2) / IgE and is the heavy chain set forth in SEQ ID NOs: 58 and 59. And contains the amino acid sequence corresponding to the light chain CDR. In certain embodiments, the anti-IL-9 (S2) / IgE-Ig protein comprises the amino acid sequences corresponding to the heavy and light chain variable regions set forth in SEQ ID NOs: 58 and 59. In certain embodiments, the anti-IL-9 (S2) / IgE-Ig protein comprises the amino acid sequences corresponding to the heavy and light chains set forth in SEQ ID NOs: 58 and 59.

In certain embodiments, the DVD-Ig protein used in the methods and compositions of the present disclosure specifically binds to IgE / IL-9 (S2) and is the heavy chain set forth in SEQ ID NOs: 60 and 61. And contains the amino acid sequence corresponding to the light chain CDR. In certain embodiments, the anti-IgE / IL-9 (S2) -Ig protein comprises the amino acid sequences corresponding to the heavy and light chain variable regions set forth in SEQ ID NOs: 60 and 61. In certain embodiments, the anti-IgE / IL-9 (S2) -Ig protein comprises the amino acid sequences corresponding to the heavy and light chains set forth in SEQ ID NOs: 60 and 61.

In certain embodiments, the DVD-Ig proteins used in the methods and compositions of the present disclosure specifically bind to TNF / IL-17 and are heavy and light chains set forth in SEQ ID NOs: 62 and 63. Contains the amino acid sequence corresponding to the CDR. In certain embodiments, the anti-TNF / IL-17-Ig protein comprises the amino acid sequences corresponding to the heavy and light chain variable regions set forth in SEQ ID NOs: 62 and 63. In certain embodiments, the anti-TNF / IL-17-Ig protein comprises the amino acid sequences corresponding to the heavy and light chains set forth in SEQ ID NOs: 62 and 63.

In certain embodiments, the DVD-Ig protein used in the methods and compositions of the present disclosure specifically binds to TNF / PGE2 to the heavy and light chain CDRs set forth in SEQ ID NOs: 64 and 65. Contains the corresponding amino acid sequence. In certain embodiments, the anti-TNF / PGE2-Ig protein comprises the amino acid sequences corresponding to the heavy and light chain variable regions set forth in SEQ ID NOs: 64 and 65. In certain embodiments, the anti-TNF / PGE2-Ig protein comprises the amino acid sequences corresponding to the heavy and light chains set forth in SEQ ID NOs: 64 and 65.

In certain embodiments, the DVD-Ig protein used in the methods and compositions of the present disclosure specifically binds IL-1α / IL-1β and the heavy chains and heavy chains set forth in SEQ ID NOs: 66 and 67 and Contains the amino acid sequence corresponding to the light chain CDR. In certain embodiments, the anti-IL-1α / IL-1β-Ig protein comprises the amino acid sequences corresponding to the heavy and light chain variable regions set forth in SEQ ID NOs: 66 and 67. In certain embodiments, the anti-IL-1α / IL-1β-Ig protein comprises the amino acid sequences corresponding to the heavy and light chains set forth in SEQ ID NOs: 66 and 67.

In certain embodiments, the DVD-Ig protein used in the methods and compositions of the present disclosure specifically binds to PLL4 / VEGF into the heavy and light chain CDRs set forth in SEQ ID NOs: 28 and 29. Contains the corresponding amino acid sequence. In certain embodiments, the anti-DLL4 / VEGF-Ig protein comprises the amino acid sequences corresponding to the heavy and light chain variable regions set forth in SEQ ID NOs: 28 and 29. In certain embodiments, the anti-DLL4 / VEGF-Ig protein comprises the amino acid sequences corresponding to the heavy and light chains set forth in SEQ ID NOs: 28 and 29.

In certain embodiments, the DVD-Ig protein used in the methods and compositions of the present disclosure specifically binds to PLL4 / VEGF into the heavy and light chain CDRs set forth in SEQ ID NOs: 72 and 73. Contains the corresponding amino acid sequence. In certain embodiments, the anti-DLL4 / VEGF-Ig protein comprises the amino acid sequences corresponding to the heavy and light chain variable regions set forth in SEQ ID NOs: 72 and 73. In certain embodiments, the anti-DLL4 / VEGF-Ig protein comprises the amino acid sequences corresponding to the heavy and light chains set forth in SEQ ID NOs: 72 and 73.

In certain embodiments, the DVD-Ig protein used in the methods and compositions of the present disclosure specifically binds IL12 / IL18 to the heavy and light chain CDRs set forth in SEQ ID NOs: 70 and 71. Contains the corresponding amino acid sequence. In certain embodiments, the anti-IL12 / IL18-Ig protein comprises the amino acid sequences corresponding to the heavy and light chain variable regions set forth in SEQ ID NOs: 70 and 71. In certain embodiments, the anti-IL12 / IL18-Ig protein comprises the amino acid sequences corresponding to the heavy and light chains set forth in SEQ ID NOs: 70 and 71.

D. Expression of DVD-Ig Protein The DVD-Ig protein of the present disclosure may be produced by any of several techniques known in the art. For example, expression from a host cell in which an expression vector (s) encoding a DVD heavy chain and a DVD light chain are transfected into the host cell by standard techniques. Various forms of the term "transfection" are commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. It is intended to embrace the wide variety of techniques used.

Mammalian host cells for expressing the recombinant antibodies of the present disclosure include Chinese hamster ovary (CHO cells) (dhfr-CHO cells, Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77: 4216- 4220, eg, used with DHFR selectable markers, as described in Kaufman and Sharp (1982) J. Mol. Biol. 159: 601-621) and DG44 or DUXB11 cells (Urlab et al. (1986)). Some. Cell Molec. Genet. 12: 555, Haynes et al. (1983) Nuc. Acid. Res. 11: 687-706, Lau et al. (1984) Mol. Cell. Biol. 4: 1469-1475),. NS0 myeloma cells, monkey kidney strains (eg, CVI and COS such as COS 7 cells), SP2 cells, human embryo-derived kidney (HEK) cells such as HEK-293 cells, Chinese hamster fibroblasts (eg, R1610), Human cervical cancer (eg HELA), mouse fibroblasts (eg BALBc / 3T3), mouse myeloma (P3x63-Ag3.653, NS0, SP2 / O), hamster kidney strain (eg HAK), mouse L Includes cells (eg L-929), human lymphocytes (eg RAJI), human kidneys (eg 293 and 293T). The host cell line is typically (eg, from BD Biosciences, Lexington, Ky., Promega, Madison, Wis., Life Technologies, Gaithersburg, Md.) Or from American Type Culture (American Type , Commercially available.

When a recombinant expression vector encoding the DVD-Ig protein is introduced into a mammalian host cell, the DVD-Ig protein causes the host cell to express the DVD-Ig protein in the host cell or to grow the host cell. It is produced by culturing for a period of time sufficient to allow the secretion of DVD-Ig protein into the culture medium. DVD-Ig protein is recovered from culture medium using standard protein purification methods.

In an exemplary system for recombinant expression of the DVD-Ig protein, recombinant expression vectors encoding both the DVD-Ig heavy chain and the DVD-Ig light chain are expressed in dhfr-CHO cells by calcium phosphate mediated transfection. Introduced in. Within the recombinant expression vector, the DVD-Ig heavy and light chain cDNAs are each operably linked to the CMV enhancer / AdMLP promoter regulatory element to drive high levels of transcription of the cDNA. The recombinant expression vector also carries a cDNA encoding DHFR, which allows the selection of vector-transfected CHO cells using methotrexate selection / amplification. Selected transformant host cells are cultured to allow expression of DVD-Ig heavy and light chains, and intact DVD-Ig protein is recovered from the culture medium. Recombinant expression vectors are prepared using standard molecular biology techniques, the host cells are transfected, the transformants are selected, the host cells are cultured, and the DVD-Ig protein is recovered from the culture medium. Still further, the disclosure provides a method of synthesizing a DVD-Ig protein of the present disclosure by culturing the host cells of the present disclosure in a suitable culture medium until the DVD-Ig protein of the present disclosure is synthesized. The method can further comprise isolating the DVD-Ig protein from the culture medium. An important feature of the DVD-Ig protein is that it can be produced and purified in the same manner as for conventional antibodies.

E. Methods for Identifying Aqueous Stable DVD-Ig (AS-DVD-Ig) Proteins and Freeze-Dried Stable DVD-Ig (LS-DVD-Ig) Proteins Many DVD-Ig proteins are unstable and aggregate While certain subsets of DVD-Ig proteins (referred to as AS-DVD-Ig proteins and LS-DVD-Ig proteins) are stable in aqueous formulations, even at high concentrations. That is an unexpected and surprising finding. In addition, while the majority of DVD-Ig proteins have been found to be unstable in the lyophilized state, certain subsets of DVD-Ig proteins (referred to as LS-DVD-Ig proteins). Is stable and can be successfully lyophilized using the formulations of the present disclosure. Notably, the DVD-Ig protein identified as the AS-DVD-Ig protein is also LS-DVD, considering that the AS-DVD-Ig protein is generally more stable than the LS-DVD-Ig protein. -It is also an Ig protein. In addition, the non-AS-DVD Ig protein may be the LS DVD-Ig protein. Discrimination between the two subpopulations may be based on the level of aggregation, freezing / thawing properties, or monomer content after storage, as described in the assay below. In general, an increase in aggregation indicates a decrease in monomer content.

Therefore, in certain embodiments, the present disclosure includes methods for distinguishing between AS-DVD-Ig proteins and non-AS-DVD-Ig proteins. The disclosure also includes methods for distinguishing between LS-DVD-Ig proteins and non-LS-DVD-Ig proteins. In another alternative form, the present disclosure provides a method for distinguishing the AS-DVD-Ig protein from the LS-DVD-Ig protein. After identification, the AS-DVD-Ig protein and the LS-DVD-Ig protein can be successfully formulated in the compositions of the present disclosure, while the non-AS-DVD-Ig protein and the non-LS-DVD-Ig protein , It cannot remain stable in such formulations and is prone to monomer content aggregation and / or loss. In general, an increase in aggregation indicates a decrease in monomer content.

In certain embodiments, the freeze / thaw (F / T) (eg, -80 ° C / 30 ° C) test is used to make a DVD-Ig protein that is an AS-DVD-Ig protein or an LS-DVD-Ig protein. May be specified. Such a method depends on determining the percentage of high molecular weight (HMW) species in solution with a high concentration of DVD-Ig protein (eg, 100 mg / mL). In one embodiment, the AS-DVD-Ig protein or LS-DVD-Ig protein has a 1% or less increase (aggregate) (eg, aggregate) of high molecular weight (HMW) species when determined by SEC after the F / T cycle. It is defined as a DVD-Ig protein that exhibits less than 0.5% aggregate), or less than 1% increase (eg, 0.5%) in relative% monomeric change. The F / T cycle is to freeze the DVD-Ig protein solution, thaw the solution, iterate at will, and test the thawed solution for aggregation levels, eg, using SEC analysis. ,including. Freezing / thawing tests may be performed on solutions containing DVD-Ig protein at concentrations of about 1 mg / mL to about 10 mg / mL. In certain embodiments, the F / T test comprises measuring aggregation or changes in monomer content by SEC after four freezing / thawing cycles at about 30 ° C to -80 ° C. A DVD-Ig protein will be considered non-LS DVD Ig if the test results in an increase of more than about 1% in the change in aggregate relative percent or monomer relative percent after 4 cycles.

In certain embodiments, the unfolding temperature of the DVD-Ig protein may be used to determine if the DVD-Ig protein is an AS-DVD-Ig protein or an LS-DVD-Ig protein. As described in FIG. 4, DVD-Ig proteins exhibiting unfolding temperatures below about 45 ° C. are generally not characterized as AS-DVD-Ig proteins or LS-DVD-Ig proteins (see FIG. 4). Described "A"). In certain other embodiments, the DVD-Ig protein exhibiting an unfolding temperature of about 45-50 ° C. is unlikely to be an AS-DVD-Ig protein, but can be an LS-DVD-Ig protein. (“B” as shown in FIG. 4). In certain other embodiments, DVD-Ig proteins exhibiting unfolding temperatures above 50 ° C. are generally considered AS-DVD-Ig proteins and LS-DVD-Ig proteins (as described in FIG. 4). "C"). Tests for determining the DVD-Ig protein unfolding temperature are known in the art (see also Example 1 describing thermodynamic tests) and are generally about 5 and about 7, For example, the pH is about 5.5 to about 6.5.

A storage test of the solution can be performed to determine if the DVD-Ig protein is an AS-DVD-Ig protein or an LS-DVD-Ig. For example, the storage test may be performed at 5 ° C. or 40 ° C. for 14-21 days with DVD-Ig protein in solution at a concentration ranging from 1 to 100 mg / mL, eg, about 50-100 mg / mL.

Testing at temperatures above ambient temperature, such as 40 ° C, is often referred to as acceleration conditions. In certain embodiments, accelerated storage conditions include storing the DVD-Ig protein at 40 ° C. in the absence of light. In certain embodiments, the test is based on the DVD-Ig protein aggregation level of the solution at high temperature (eg, 40 ° C.) and high concentration (eg, 50 mg / mL) as determined by SEC. For example, the DVD-Ig protein is formulated in an aqueous formulation using citrate phosphate buffer or histidine buffer at a concentration of at least about 50 mg / mL, eg, 50-100 mg / mL, under accelerated conditions. It may be stored in. Acceleration conditions may include temperatures above room temperature, such as storage temperatures of about 35 to about 45 degrees Celsius (C), for long periods of time, such as about 10 to about 21 days. In certain embodiments, the accelerated storage conditions used to screen for AS-DVD-Ig protein or LS-DVD-Ig protein are 50 mg / mL or higher, eg, about 60 mg / mL or 50-100 mg / mL. DVD-Ig protein concentration, stored at a temperature of 40 ° C. for 14 days. After an accelerated storage test at a concentration of 50 mg / mL or higher, eg, 50-100 mg / mL, the solution may be tested for signs of DVD-Ig protein aggregation or changes in monomer content.

In certain embodiments, a buffered solution having a pH of 5.0 to 6.5, such as a pH of about 6, and a concentration of about 50 mg / mL to about 100 mg / mL (eg, histidine or citrate /). DVD-Ig proteins may be tested in phosphate buffer) to determine if they are AS-DVD-Ig proteins or LS-DVD-Ig proteins.

Notably, lower levels of DVD-Ig protein concentration (eg, 1 mg / mL) may also be used to test the protein, in which case lower levels of aggregation will result in AS-DVD-Ig protein or As expected for LS-DVD-Ig. For example, the AS-DVD-Ig protein is a DVD-Ig with up to 3% aggregation after 21 days when stored at a concentration of 1 mg / mL in an aqueous formulation at about 40 ° C.

Protein aggregation may be determined according to methods known in the art, including but not limited to size exclusion chromatography (SEC). In general, an increase in aggregation indicates a decrease in monomer content, and this decrease in monomer content can also be determined using SEC analysis.

In certain embodiments, the DVD-Ig protein is at a concentration of 1-100 mg / mL, preferably about 50 mg / mL to about 100 mg / mL, when the solution is determined by size exclusion chromatography (SEC) analysis. If it has less than 10% aggregation of DVD-Ig protein after accelerated storage, it is considered AS-DVD-Ig protein. In certain embodiments, the DVD-Ig protein aggregates less than 6% of the DVD-Ig protein after accelerated storage at a concentration of about 50 mg / mL to about 100 mg / mL when the solution is determined by SEC analysis. Is considered to be an AS-DVD-Ig protein. In certain embodiments, the DVD-Ig protein is less than 10%, as an alternative, after accelerated storage at a concentration of about 50 mg / mL to about 100 mg / mL, when the DVD-Ig protein is determined by SEC analysis. If it has less than%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% aggregation, it is considered an AS-DVD-Ig protein. .. In certain embodiments, the AS-DVD-Ig protein is defined as a DVD-Ig with less than 8% aggregation after 14 days of accelerated storage (eg, at about 40 ° C.). In certain embodiments, the AS-DVD-Ig protein is defined as a DVD-Ig having no more than 6% aggregation after 14 days of accelerated storage (eg, at about 40 ° C.). In a further embodiment, the AS-DVD-Ig protein is defined as a DVD-Ig having less than 5% aggregation after 14 days of accelerated storage (eg, at about 40 ° C.). In certain embodiments, the AS-DVD-Ig protein has less than 10% aggregation after storage at about 40 ° C. for 21 days at a concentration of 100 mg / mL in an aqueous formulation, or 50 mg / mL or more in an aqueous formulation. Defined as DVD-Ig with less than 10% aggregation after 14 days of accelerated storage at about 40 ° C. when formulated at the concentration of.

In certain embodiments, the AS-DVD-Ig protein is a DVD-Ig protein, or aqueous, which has less than 10% aggregation after storage in an aqueous formulation at a concentration of 100 mg / mL at about 40 ° C. for 21 days. It is defined as a DVD-Ig protein with less than 1% aggregation after storage in the formulation at a concentration of 100 mg / mL at about 5 ° C. for 21 days. Alternatively, AS-DVD-Ig protein is stored at a concentration of 1 mg / mL in an aqueous formulation at about 5 ° C. for 21 days, followed by agglutination of 1.5% or less, or about 40 at a concentration of 1 mg / mL in an aqueous formulation. A DVD-Ig protein having less than 3% aggregation after storage at ° C. for 21 days. In certain embodiments, the AS-DVD-Ig protein, when formulated in aqueous formulations at concentrations above 50 mg / mL, has less than 10% aggregates after 14 days of accelerated storage at about 40 ° C. It is defined as a DVD-Ig protein that is formed. In certain embodiments, the AS-DVD-Ig protein is defined as a DVD-Ig protein that has less than 10% aggregation after 14 days of accelerated storage (eg, at about 40 ° C.). In certain embodiments, the AS-DVD-Ig protein is defined as a DVD-Ig protein that has less than 8% aggregation after 14 days of accelerated storage (eg, at about 40 ° C.). In certain embodiments, the AS-DVD-Ig protein is defined as a DVD-Ig protein with up to 6% aggregation after 14 days of accelerated storage (eg, at about 40 ° C.). In a further embodiment, the AS-DVD-Ig protein is defined as a DVD-Ig protein with less than 5% aggregation after 14 days of accelerated storage (eg, at about 40 ° C.). Aggregation can be determined according to methods known in the art, including but not limited to size exclusion chromatography (SEC). In certain embodiments, accelerated storage conditions include storing the DVD-Ig protein at 40 ° C. in the absence of light. DVD-Ig proteins may be tested in aqueous formulations containing citrate and phosphate buffers, or histidine buffers. In certain embodiments, the AS-DVD-Ig protein, as determined by SEC analysis, has less than 10% aggregation after 21 days of accelerated storage at about 40 ° C., where the AS-DVD-Ig protein. Is formulated at a concentration of 50-100 mg / mL in citrate phosphate buffer or histidine buffer in aqueous formulations. In certain embodiments, the AS-DVD-Ig protein has less than 6% aggregation, as determined by SEC analysis, after 14 days of accelerated storage at about 40 ° C., where the AS-DVD-Ig protein. Is formulated at a concentration of at least 50 mg / mL in citrate phosphate buffer or histidine buffer in an aqueous formulation.

Percentage of agglomeration may be used to determine if agglutination is present after accelerated storage, but the monomer content of the DVD-Ig protein solution may also be used. In general, an increase in percent aggregation indicates a decrease in monomer content. Alternatively, the DVD-Ig protein is a DVD-Ig protein of 50 mg / mL or more at a pH of about 5.0 to about 6.5, for example about 5.5 to about 6.0, in 15 mM histidine. In a solution having a protein concentration, it has a monomer loss of 6% or less after 14 days at 40 ° C. (determined by SEC) or a monomer loss of 3% or less after 7 days at 40 ° C. (determined by SEC). If so, it may be considered as an AS-DVD-Ig protein. Any test in which the monomer content includes, but is not limited to, storage at 40 ° C. and / or at a pH of about 5.0 to about 6.5, eg, about 5.5 to about 6.0. It may be used under conditions.

In certain embodiments, the AS-DVD-Ig protein has a monomer relative (rel.) Peak of about 10% or less after storage in an aqueous formulation at a concentration of about 100 mg / mL at about 40 ° C. for 21 days. DVD-Ig protein with area variation, or about 1% or less after storage at a concentration of about 100 mg / mL and about 5.5 to about 6.5 pH at about 5 ° C. for 21 days in an aqueous formulation. A DVD-Ig protein having a monomeric relative (rel.) Peak area variation. Alternatively, the AS-DVD-Ig protein has a monomer relative (rel.) Peak area change of about 1.5% or less after storage in an aqueous formulation at a concentration of about 1 mg / mL at about 5 ° C. for 21 days. Or, in an aqueous formulation, a monomer relative (rel.) Peak area change of about 3% or less after storage at a concentration of 1 mg / mL and at about 5.5 to about 6.5 pH at about 40 ° C. for 21 days. It is a DVD-Ig protein having. In certain embodiments, the AS-DVD-Ig protein is about 40 ° C. when formulated in an aqueous formulation at a concentration greater than about 50 mg / mL and at a pH of about 5.5 to about 6.5. It is defined as a DVD-Ig protein with a monomeric change in relative (rel.) Peak area of less than about 10% after accelerated storage after 14 days. In certain embodiments, the AS-DVD-Ig protein is formulated in an aqueous formulation at a concentration greater than 60 mg / mL and at a pH of 5.5-6.5 (eg, about 40 ° C.). Defined as a DVD-Ig protein with less than 8% monomeric relative (rel.) Peak area change after 14 days of accelerated storage. In certain embodiments, the AS-DVD-Ig protein has a monomer-relative (rel.) Peak area change of 6% or less after 14 days of accelerated storage (eg, at about 40 ° C.), DVD- Defined as Ig protein. In a further embodiment, the AS-DVD-Ig protein has a monomer relative (rel.) Peak area change of less than 5% after 14 days of accelerated storage (eg, at about 40 ° C.). Is defined as.

In certain embodiments, the AS-DVD-Ig protein has a monomer relative (rel.) Peak area change of 10% or less after 21 days of accelerated storage at about 40 ° C. as determined by SEC analysis. And here the AS-DVD-Ig protein is a citrate phosphate buffer or histidine buffer having a pH of about 5.0 to about 6.5, eg, about 5.5 to about 6.0, in an aqueous formulation. It is formulated in liquid at a concentration of 50-100 mg / mL. In certain embodiments, the AS-DVD-Ig protein has less than 6% monomeric relative (rel.) Peak area change after 14 days of accelerated storage at about 40 ° C. as determined by SEC analysis. The AS-DVD-Ig protein is then formulated in an aqueous formulation in citrate phosphate buffer or histidine buffer at a concentration of at least 50 mg / mL.

In another alternative form, the AS-DVD-Ig protein, when determined by SEC, has the aggregation stability of the solution at low temperature (eg, 5 ° C.) and high concentration (eg, 50 mg / mL) of DVD-Ig. / Or specified based on monomer content. For example, a solution containing 50 mg / mL AS-DVD-Ig protein at a pH of about 5.0 to about 6.5, eg, about 5.5 to about 6.0, in 15 mM histidine is 5 ° C. After 7 days (determined by SEC), it may have a monomer loss of 1% or less (determined by SEC). In another example, a solution containing 50 mg / mL AS-DVD-Ig protein in 15 mM histidine at a pH of about 5.0 to about 6.5, eg, about 5.5 to about 6.0. After 14 days at 5 ° C., it may have a monomer loss of 2% or less. Alternatively, AS-DVD-Ig is stored at a concentration of 100 mg / mL in an aqueous formulation at about 5 ° C. for 21 days, followed by agglutination of 1% or less, or 21 at a concentration of 1 mg / mL in an aqueous formulation at about 5 ° C. After storage for days, it has less than 1.5% aggregation. In certain embodiments, the monomer loss is determined at a pH of about 5.0 to about 6.5, eg, about 5.5 to about 6.0.

In certain embodiments, the test solution conditions described herein also contain 0.02% (w / v) sodium azide as a bacteriostatic agent.

In certain embodiments, the DVD-Ig protein will be associated with the LS-DVD-Ig protein if the solution has less than 15% aggregation of the DVD-Ig protein as determined by size exclusion chromatography (SEC) analysis. Be considered. In certain embodiments, the LS-DVD-Ig protein is less than 15%, as an alternative less than 14%, less than 13%, less than 12%, less than 11% when the DVD-Ig protein is determined by SEC analysis. If it has aggregation, it is considered an LS-DVD-Ig protein.

In certain embodiments, the LS-DVD-Ig protein is 15% after accelerated storage, eg, accelerated storage at about 40 ° C. for 14 days, when formulated at concentrations above 50 mg / mL in aqueous formulations. It is defined as the formation of less than agglomerates. In certain embodiments, the LS-DVD-Ig protein has no more than 15% aggregation after 14 days of accelerated storage, eg, at 40 ° C. In certain embodiments, the LS-DVD-Ig protein has less than 14% aggregation, for example, after 14 days of accelerated storage at about 40 ° C. In a further embodiment, the LS-DVD-Ig protein has less than 13% aggregation after 14 days of accelerated storage, for example at about 40 ° C. Alternatively, the LS-DVD-Ig protein is defined as a DVD-Ig protein with less than 1% aggregation after 4 freeze-thaw cycles. DVD-Ig proteins may be tested in aqueous formulations containing citrate and phosphate buffers, or histidine buffers. In certain embodiments, the LS-DVD-Ig protein, as determined by SEC analysis, has no more than 15% aggregation after 14 days of accelerated storage at about 40 ° C., where the LS-DVD-Ig protein. Is formulated at a concentration of at least 50 mg / mL in citrate phosphate buffer in an aqueous formulation. As mentioned above, aggregation can be determined according to methods known in the art, including but not limited to SEC.

In certain embodiments, the LS-DVD-Ig protein is in an aqueous formulation at a concentration of about 50-100 mg / mL and has a pH of about 5.0 to about 6.5, such as about 5.5 to about 6. After 21 days of accelerated storage, eg, at about 40 ° C., when formulated at 0, greater than 10% monomeric relative (rel.) Peak area changes are observed. In certain embodiments, the LS-DVD-Ig protein has a monomer relative (rel.) Peak area change of 50% or less after 14 days of accelerated storage at about 40 ° C. as determined by SEC analysis. Here, the LS-DVD-Ig protein is formulated at a concentration of at least 50 mg / mL in citrate phosphate buffer in an aqueous formulation. In certain embodiments, the LS-DVD-Ig protein is in an aqueous formulation at a pH of about 5.0 to about 6.5, eg, about 5.5 to about 6.0, at concentrations above 100 mg / mL. When formulated in, it has a monomer relative (rel.) Peak area change of 20% or less after 21 days of accelerated storage at about 40 ° C. In certain embodiments, the LS-DVD-Ig protein has a monomer-relative (rel.) Peak area change of less than 18%, for example, after 14 days of accelerated storage at about 40 ° C. In a further embodiment, the LS-DVD-Ig protein has a monomer relative (rel.) Peak area change of less than 13%, for example, at about 40 ° C. after 14 days of accelerated storage. Alternatively, the LS-DVD-Ig protein is defined as a DVD-Ig protein with a monomer relative (rel.) Peak area change of 1% or less after 4 freeze-thaw cycles. Alternatively, the LS-DVD-Ig protein is prepared in an aqueous solution at the most stable pH at a concentration of 1-100 mg / mL, eg, about 1-10 mg / mL or about 50-100 mg / mL, at about 25 ° C. for 7 days. Later, it is defined as a DVD-Ig protein with a monomer relative (rel.) Peak area change of 4% or less. Alternatively, the LS-DVD-Ig protein is defined as a DVD-Ig protein with a monomer relative (rel.) Peak area change of 1% or less after 7 days at about 5 ° C. in the most stable aqueous solution. To.

Notably, the assays described herein used to determine if a DVD-Ig protein is an AS-DVD-Ig protein or an LS-DVD-Ig protein are exclusive. That is, for example, AS-DVD-Ig may be characterized as having an unfolding temperature of at least 50 ° C. and at a concentration of about 50 to about 100 mg / mL in an aqueous buffer formulation. After storage for 21 days at about 5 ° C. at a pH of about 5.5 to about 6.5, there may be a monomer relative peak area change of about 1% or less. In certain embodiments, the AS-DVD-Ig protein has a monomer relative (rel.) Peak area change of 10% or less after 21 days of accelerated storage at about 40 ° C. as determined by SEC analysis. And have an unfolding temperature of at least 50 ° C.

Once the DVD-Ig protein has been identified as an AS-DVD-Ig protein or LS-DVD-Ig protein according to the tests described above, the AS-DVD-Ig protein or LS-DVD-Ig protein is stable. It can be formulated. Further identification of the AS-DVD-Ig protein and the LS-DVD-Ig protein is described below in Example 4.

III. Aqueous Stable Double Variable Domain Immunoglobulin (AS-DVD-Ig) Formulations of the Present Disclosure The present disclosure provides stable aqueous formulations containing the AS-DVD-Ig protein. The disclosure features an aqueous formulation that has improved properties compared to formulations recognized in the art in that AS-DVD-Ig protein can be stably formulated even at high concentrations. And.

Thus, in the present disclosure, at least in part, a subpopulation of DVD-Ig proteins has a pH of about 4.5 to about 7.5 and contains buffers, detergents, and / or polyols. It is also based on the discovery that it can be stably formulated in aqueous formulations. These "aqueous stable DVD-Ig proteins" are referred to as AS-DVD-Ig proteins and can be identified using an accelerated storage assay, where the DVD-Ig protein is 50 mg / mL, eg, 50 mg / mL. , 50 mg / mL to greater than 100 mg / mL, formulated in liquid form (see also Section II.E above).

In certain embodiments, the aqueous formulations of the present disclosure have a pH of about 4.5 to about 7.5. As described in the actual examples, pH was found to have an effect on the stability of the AS-DVD-Ig protein in buffered formulations. In certain embodiments, the pH of the formulation containing the AS-DVD-Ig protein ranges from about 4.5 to about 7.5, and as an alternative, the pH of the AS-DVD-Ig protein formulation is about. The pH is in the range of 5.0 to about 7.0, and as an alternative the pH may be in the range of about 5 to about 6.5, and as an alternative the pH of the formulation is about 5.5 to about 6.5. It may be in the range of. In a further embodiment, the pH is in the range of about 5.8 to about 6.2. An intermediate range of pH values, such as about 5.6 to about 6.4, is also intended to be part of the present disclosure. It is also intended to include a range of values using any combination of the above values as the upper / lower limit, eg, a pH range of about 5.5 to about 6.2. In certain embodiments, the pH of the formulations of the present disclosure is about 6.0.

In certain embodiments, the aqueous formulations of the present disclosure include AS-DVD-Ig protein and buffer. Examples of buffers that can be used in the formulations of the present disclosure include, but are not limited to, acetates, histidines, glycines, arginines, phosphates, tris, and citrates. The molar concentration of the buffer used in the formulations of the present disclosure may range from about 1 to about 50 mM. In certain embodiments, the aqueous formulations of the present disclosure have a buffer solution at a molar concentration of about 5 to about 50 mM. Alternatively, the molar concentration of the buffer is about 10 to about 20 mM.

In certain embodiments of the present disclosure, the buffer system is at a pH of about 4.5 to about 7.5, such as a pH of about 5 to about 7, or a pH of about 5.5 to about 6.5. , About 1 to about 200 mM histidine (eg, about 2 to about 100 mM, about 5 to about 70 mM, about 5 to about 60 mM, about 5 to about 50 mM, about 10 to about 40 mM, about 10 to about 30 mM, or about 10). ~ About 20 mM). In certain embodiments, the buffer system of the present disclosure is at a pH of about 4.5 to about 7.5, such as a pH of about 5 to about 7, or a pH of about 5.5 to about 6.5. , Containing about 15 mM histidine.

In certain embodiments, the buffer system comprises about 1 to about 50 mM (eg, about 5 to about 40 mM) of glycine at a pH of about 4.5 to about 7.5. In certain embodiments, the buffer system comprises glycine at a concentration of about 20 mM. In a further specific embodiment, the buffer system is at a pH of about 4.5 to about 7.5, such as a pH of about 5 to about 7, a pH of about 5.5 to about 6.5, and about 20 mM. It contains a concentration of glycine and a concentration of about 20 to about 30 mg / mL, eg, about 26 mg / mL of glycerol.

In certain embodiments, the buffer system is at a pH of about 4.5 to about 7.5, such as a pH of about 5 to about 7, or a pH of about 5.5 to about 6.5, about 1 Includes ~ about 50 mM acetate (eg, about 5 to about 50 mM, about 2 to about 40 mM, about 5 to about 30 mM, or about 2 to about 15 mM). In certain embodiments, the buffer system comprises acetate at a concentration of about 2 to about 15 mM.

In certain embodiments of the present disclosure, the buffer system is at a pH of about 4.5 to about 7.5, such as a pH of about 5 to about 7, or a pH of about 5.5 to about 6.5. , Approximately 1 to about 50 mM (eg, about 5 to about 50 mM, about 2 to about 40 mM, about 5 to about 30 mM, or about 2 to about 15 mM) of arginine. In certain embodiments, the buffer system comprises arginine at a concentration of about 15 mM.

In yet another embodiment of the present disclosure, the buffer system is at a pH of about 4.5 to about 7.5, such as a pH of about 5 to about 7, or a pH of about 5.5 to about 6.5. , Includes about 1 to about 50 mM (eg, about 5 to about 50 mM) citrate. In certain embodiments, the buffer system comprises citrate at a concentration of about 15 mM.

In yet another embodiment of the present disclosure, the buffer system is at a pH of about 4.5 to about 7.5, such as a pH of about 5 to about 7, or a pH of about 5.5 to about 6.5. , Includes about 1 to about 50 mM (eg, about 5 to about 50 mM) phosphate. In certain embodiments, the buffer system comprises phosphate at a concentration of about 10 mM. In one embodiment, the buffer system comprises phosphate at a concentration of about 10 mM and sodium chloride at a concentration of about 125 mM.

In certain embodiments, the buffer system is at a pH of about 4.5 to about 7.5, such as a pH of about 5 to about 7, or a pH of about 5.5 to about 6.5, about 1 Includes ~ about 50 mM (eg, about 5 to about 50 mM) Tris. In certain embodiments, the buffer system comprises Tris at a concentration of about 2 to about 10 mM.

Still in another embodiment, the buffer system comprises phosphates and citrates, such as phosphates, at concentrations of about 1 to about 50 mM (eg, about 5 to about 50 mM, about 5 to about 10 mM). For example, sodium hydrogen phosphate), as well as citrate (citric acid) at a concentration of about 1 to about 50 mM (eg, about 5 to about 10 mM). In certain embodiments, the buffer system comprises phosphate at a concentration of about 5 mM and citrate (citric acid) at a concentration of about 5 mM. In certain embodiments, the buffer system comprises phosphate at a concentration of about 10 mM and citrate (citric acid) at a concentration of about 10 mM.

In addition to the buffer, polyol may be added to the aqueous formulation, eg, for additional stability. The polyol may be added to the formulation in an amount that may vary with respect to the desired isotonicity of the formulation. In certain embodiments, the aqueous formulation is isotonic.

Examples of polyols that can be used in the aqueous formulations of the present disclosure include, but are limited to, sucrose, fructose, mannose, maltose, lactose, arabinose, xylose, ribose, lambose, galactose, and glucose. Not done. Non-reducing sugars include sucrose, trehalose, sorbose, melesitose, raffinose, mannitol, xylitol, erythritol, threitol, sorbitol, and glycerol. The amount of polyol added can also vary with respect to the molecular weight of the polyol. For example, lower amounts of monosaccharides (eg mannitol) may be added as compared to disaccharides (eg trehalose).

In certain embodiments, the concentration of polyols such as sorbitol is from about 30 to about 50 mg / mL. In one embodiment, the composition comprises from about 20 to about 60 mg / mL sorbitol, from about 25 to about 55 mg / mL, from about 30 to about 50 mg / mL, from about 35 to about 45 mg / mL, eg, about 33. It is in the range of ~ about 48 mg / mL sorbitol.

In certain embodiments, sucrose has a concentration of about 70-about 90 mg / mL. In certain embodiments, the composition comprises from about 60 to about 100 mg / mL sucrose, from about 65 to about 95 mg / mL, from about 70 to about 90 mg / mL, from about 75 to about 85 mg / mL, eg, It ranges from about 72 to about 84 mg / mL sucrose.

In certain embodiments, the polyol is mannitol. In certain embodiments, the composition comprises about 10 to about 100 mg / mL, or about 20 to about 80, about 20 to about 70, about 30 to about 60, about 30 to about 50 mg / mL of mannitol, eg. , About 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 mg / mL.

In certain embodiments, the aqueous formulation of the present disclosure comprises AS-DVD-Ig, a buffer having a molar concentration of about 5 to about 50 mM, and a polyol, which formulation is about 4.5 to about 7. It has a pH of 5.

In addition to the buffer, surfactants may be added to the aqueous formulation, eg, for additional stability. Examples of surfactants include nonionic detergents such as polysorbate (eg, polysorbate 20, 80) or poloxamer (eg, poloxamer 188). In certain embodiments, the amount of detergent added reduces the aggregation of the formulated AS-DVD-Ig protein and / or minimizes the formation of microparticles in the formulation and / or The amount is such that adsorption is reduced.

In certain embodiments, the aqueous formulation contains the detergent polysorbate 80 or Tween 80. Tween 80 is a term used to describe polyoxyethylene (20) sorbitan monooleate. In certain embodiments, the formulation comprises about 0.001 to about 1% polysorbate 80, or about 0.005 and about 0.05% polysorbate 80, such as about 0.001%, about 0.005. , About 0.01%, about 0.05%, or about 0.1% of polysorbate 80. In certain embodiments, about 0.01% polysorbate 80 is found in the formulations of the present disclosure.

In certain embodiments, the aqueous formulation of the present disclosure comprises AS-DVD-Ig, a buffer having a molar concentration of about 5 to about 50 mM, and a surfactant, which formulation is about 4.5 to about. It has a pH of 7.5. In certain embodiments, the surfactant is polysorbate, such as polysorbate 80 or polysorbate 20. In certain embodiments, the polysorbate has a concentration of about 0.005% to about 0.02%.

In certain embodiments, the aqueous formulation of the present disclosure comprises AS-DVD-Ig, a buffer solution having a molar concentration of about 5 to about 50 mM, a surfactant, and a polyol, which formulation is about 4.5. It has a pH of ~ about 7.5. In certain embodiments, the formulation comprises AS-DVD-Ig, buffer (eg, histidine), polysorbate, such as polysorbate 80, and sugar alcohols, such as mannitol or sorbitol. In certain embodiments, the formulation comprises AS-DVD-Ig, buffer (eg, histidine), polysorbate, such as polysorbate 80, and non-reducing sugar, such as sucrose.

One advantage of the aqueous formulations of the present disclosure is that high concentrations of AS-DVD-Ig protein can be stably maintained in aqueous solution. Thus, in certain embodiments, the formulations of the present disclosure are, for example, greater than about 10 mg / mL, greater than about 20 mg / mL, greater than about 30 mg / mL, greater than about 40 mg / mL, greater than about 50 mg / mL, and about 100 mg / mL. Over mL, over 110 mg / mL, over 120 mg / mL, over 130 mg / mL, over 140 mg / mL, over 150 mg / mL, over 160 mg / mL, over 170 mg / mL, over 180 mg / mL , Containing high protein concentrations, including protein concentrations greater than about 190 mg / mL, or greater than about 200 mg / mL.

In the various embodiments of the present disclosure, the concentrations of AS-DVD-Ig protein in aqueous formulations are about 0.1-250 mg / mL, about 0.5-220 mg / mL, about 1-210 mg / mL, about 5. ~ 200 mg / mL, about 10-195 mg / mL, about 15-190 mg / mL, about 20-185 mg / mL, about 25-180 mg / mL, about 30-175 mg / mL, about 35-170 mg / mL, about 40- 165 mg / mL, about 45-160 mg / mL, about 50-155 mg / mL, about 55-150 mg / mL, about 60-145 mg / mL, about 65-140 mg / mL, about 70-135 mg / mL, about 75-130 mg / ML, about 80-125 mg / mL, about 85-120 mg / mL, about 90-H5 mg / mL, about 95-110 mg / mL, about 95-105 mg / mL, or about 100 mg / mL. An intermediate range of concentrations listed above, such as about 31-174 mg / mL, is also intended to be part of this disclosure. For example, a range of values using any combination of the values listed above as the upper / lower bound is also intended to be included.

The present disclosure features aqueous formulations with improved properties compared to formulations recognized in the art. For example, the formulations of the present disclosure have AS-DVD-Ig protein aggregation levels of less than 7%, less than 6%, or less than 5% aggregates.

In certain embodiments, the AS-DVD-Ig proteins used in the aqueous formulations of the present disclosure have heavy and light chain sequences having the amino acid sequences set forth in SEQ ID NOs: 62 and 63, respectively, anti-TNF. / IL-17 DVD-Ig protein.

In certain embodiments, the aqueous formulation of the present disclosure comprises a weight comprising an amino acid sequence corresponding to an anti-TNF / IL-17 DVD-Ig protein (eg, heavy chain and light chain CDRs set forth in SEQ ID NOs: 62 and 63). Includes anti-TNF / IL-17 DVD-Ig proteins, including chains and light chains, or heavy and light chains comprising amino acid sequences corresponding to the heavy and light chain variable regions set forth in SEQ ID NOs: 62 and 63. Includes anti-TNF / IL-17 DVD-Ig protein, or DVD-A), histidine or acetate buffer, and sucrose or sorbitol, the formulation has a pH of about 5 to about 6, and the protein concentration is It is about 50 mg / mL or more. In a further embodiment, the aqueous formulation has a pH in the range of about 5 to about 5.5, eg, about 5.2. In a further embodiment, the aqueous formulation comprises a surfactant, eg, polysorbate. The ranges for buffers, excipients, and pH listed are described above.

In certain embodiments, the AS-DVD-Ig protein comprises an anti-IL1α / IL1β DVD-Ig protein having heavy and light chain sequences having the amino acid sequences set forth in SEQ ID NOs: 66 and 67, respectively. / IL-1β DVD-Ig.

In certain embodiments, the aqueous formulations of the present disclosure include heavy chains and amino acid sequences corresponding to the anti-IL1α / IL1β DVD-Ig proteins (eg, heavy and light chain CDRs set forth in SEQ ID NOs: 66 and 67). Anti-TL1α / IL1β DVD-Ig protein, including light chain, or anti-TNF / IL, comprising heavy and light chains comprising amino acid sequences corresponding to the heavy and light chain variable regions set forth in SEQ ID NOs: 66 and 67. Containing -17 DVD-Ig protein, or DVD-C), histidine buffer, and sucrose or sorbitol, the formulation has a pH of about 5 to about 6, and the protein concentration is at about 50 mg / mL or higher. is there. In a further embodiment, the aqueous formulation has a pH in the range of about 5 to about 5.5, eg, about 5.4. In a further embodiment, the aqueous formulation comprises a surfactant, eg, polysorbate. The ranges for buffers, excipients, and pH listed are described above.

In certain embodiments, the aqueous formulations of the present disclosure are amino acids corresponding to the anti-DLL4 / VEGF DVD-Ig proteins (eg, SEQ ID NOs: 28 and 29 or heavy and light chain CDRs set forth in SEQ ID NOs: 72 and 73. Includes an anti-DLL4 / VEGF DVD-Ig protein, including heavy and light chains comprising sequences, or amino acid sequences corresponding to heavy and light chain variable regions set forth in SEQ ID NOs: 28 and 29 or SEQ ID NOs: 72 and 73 Includes anti-DLL4 / VEGF DVD-Ig protein), including heavy and light chains, histidine buffer, and sucrose or arginine, the formulation has a pH of about 5 to about 6, and the protein concentration is about. It is from 20 mg / mL to about 50 mg / mL. In a further embodiment, the aqueous formulation has a pH in the range of about 5.5 to about 6, and in a further embodiment, the aqueous formulation is a surfactant, eg, polysorbate. The ranges for buffers, excipients, and pH listed are described above.

IV. Lyophilized and Stable Double Variable Domain Immunoglobulin (LS-DVD-Ig) Protein Formulations of the Present Disclosure The present disclosure further provides stable lyophilized formulations containing the LS-DVD-Ig protein. Thus, in the present disclosure, at least in part, a subpopulation of DVD-Ig proteins has a pH of about 4.5 to about 7.5 and contains buffers, detergents, and / or polyols. It is also based on the discovery that it can be stably formulated in lyophilized formulations. These "freeze-dried stable DVD-Ig proteins" or "LS-DVD-Ig proteins" can be identified using the accelerated storage assay (described above), where the DVD-Ig protein is 50 mg. Formulated in liquid form at concentrations greater than / mL.

The features mentioned under the lyophilized formulations of the present disclosure may refer to solutions before lyophilization, or may refer to reconstituted formulations, for example referring to liquid concentration (mg / mL). ..

In certain embodiments, the lyophilized formulations of the present disclosure have a pH of about 4.5 to about 7.5. In certain embodiments, the pH of the formulation containing the LS-DVD-Ig protein ranges from about 4.5 to about 7.5, and as an alternative, the pH of the LS-DVD-Ig protein formulation is about. The pH is in the range of 5.0 to about 7.0, and as an alternative the pH may be in the range of about 5 to about 6.5, and as an alternative the pH of the formulation is about 5.5 to about 6.5. It may be in the range of. In a further embodiment, the pH is in the range of about 5.8 to about 6.2. An intermediate range of pH values, such as about 5.6 to about 6.4, is also intended to be part of the present disclosure. It is also intended to include a range of values using any combination of the above values as the upper / lower limit, eg, a pH range of about 5.5 to about 6.2. In certain embodiments, the pH of the formulations of the present disclosure is about 6.0.

In certain embodiments, the lyophilized formulations of the present disclosure include LS-DVD-Ig protein and buffer. Examples of buffers that can be used in the formulations of the present disclosure include, but are not limited to, acetates, histidines, glycines, arginines, phosphates, tris, and citrates. The molar concentration of the buffer used in the formulations of the present disclosure may range from about 1 to about 50 mM. In certain embodiments, the aqueous formulations of the present disclosure have a buffer solution at a molar concentration of about 5 to about 50 mM. Alternatively, the molar concentration of the buffer is about 10 to about 20 mM.

In certain embodiments of the present disclosure, the buffer system is at a pH of about 4.5 to about 7.5, such as a pH of about 5 to about 7, or a pH of about 5.5 to about 6.5. , About 1 to about 200 mM histidine (eg, about 2 to about 100 mM, about 5 to about 70 mM, about 5 to about 60 mM, about 5 to about 50 mM, about 10 to about 40 mM, about 10 to about 30 mM, or about 10). ~ About 20 mM). In certain embodiments, the buffer system of the present disclosure is at a pH of about 4.5 to about 7.5, such as a pH of about 5 to about 7, or a pH of about 5.5 to about 6.5. , Containing about 15 mM histidine.

In certain embodiments, the buffer system comprises about 1 to about 50 mM (eg, about 5 to about 40 mM) of glycine at a pH of about 4.5 to about 7.5. In certain embodiments, the buffer system comprises glycine at a concentration of about 20 mM. In a further specific embodiment, the buffer system is at a pH of about 4.5 to about 7.5, such as a pH of about 5 to about 7, a pH of about 5.5 to about 6.5, and about 20 mM. It contains a concentration of glycine and a concentration of about 20 to about 30 mg / mL, eg, about 26 mg / mL of glycerol.

In certain embodiments, the buffer system is at a pH of about 4.5 to about 7.5, such as a pH of about 5 to about 7, or a pH of about 5.5 to about 6.5, about 1 Includes ~ about 50 mM acetate (eg, about 5 to about 50 mM, about 2 to about 40 mM, about 5 to about 30 mM, or about 2 to about 15 mM). In certain embodiments, the buffer system comprises acetate at a concentration of about 2 to about 15 mM.

In certain embodiments of the present disclosure, the buffer system is at a pH of about 4.5 to about 7.5, such as a pH of about 5 to about 7, or a pH of about 5.5 to about 6.5. , Approximately 1 to about 50 mM (eg, about 5 to about 50 mM, about 2 to about 40 mM, about 5 to about 30 mM, or about 2 to about 15 mM) of arginine. In certain embodiments, the buffer system comprises arginine at a concentration of about 15 mM.

In yet another embodiment of the present disclosure, the buffer system is at a pH of about 4.5 to about 7.5, such as a pH of about 5 to about 7, or a pH of about 5.5 to about 6.5. , Includes about 1 to about 50 mM (eg, about 5 to about 50 mM) citrate. In certain embodiments, the buffer system comprises citrate at a concentration of about 15 mM.

In yet another embodiment of the present disclosure, the buffer system is at a pH of about 4.5 to about 7.5, such as a pH of about 5 to about 7, or a pH of about 5.5 to about 6.5. , Includes about 1 to about 50 mM (eg, about 5 to about 50 mM) phosphate. In certain embodiments, the buffer system comprises phosphate at a concentration of about 10 mM. In one embodiment, the buffer system comprises phosphate at a concentration of about 10 mM and sodium chloride at a concentration of about 125 mM.

In certain embodiments, the buffer system is at a pH of about 4.5 to about 7.5, such as a pH of about 5 to about 7, or a pH of about 5.5 to about 6.5, about 1 Includes ~ about 50 mM (eg, about 5 to about 50 mM) Tris. In certain embodiments, the buffer system comprises Tris at a concentration of about 2 to about 10 mM.

Still in another embodiment, the buffer system comprises phosphates and citrates, such as phosphates, at concentrations of about 1 to about 50 mM (eg, about 5 to about 50 mM, about 5 to about 10 mM). For example, sodium hydrogen phosphate), as well as citrate (citric acid) at a concentration of about 1 to about 50 mM (eg, about 5 to about 10 mM). In certain embodiments, the buffer system comprises phosphate at a concentration of about 5 mM and citrate (citric acid) at a concentration of about 5 mM. In certain embodiments, the buffer system comprises phosphate at a concentration of about 10 mM and citrate (citric acid) at a concentration of about 10 mM.

In addition to the buffer, polyol may be added to the aqueous formulation, eg, for additional stability. The polyol may be added to the formulation in an amount that may vary with respect to the desired isotonicity of the formulation. In certain embodiments, the lyophilized formulation is isotonic when reconstituted.

Examples of polyols that can be used in the lyophilized formulations of the present disclosure include, but are not limited to, mannitol, sucrose, trehalose, and raffinose. The amount of polyol added can also vary with respect to the molecular weight of the polyol. For example, lower amounts of monosaccharides (eg mannitol) may be added as compared to disaccharides (eg trehalose).

In certain embodiments, the concentration of polyols such as sorbitol is from about 30 to about 50 mg / mL. In one embodiment, the composition comprises from about 20 to about 60 mg / mL sorbitol, from about 25 to about 55 mg / mL, from about 30 to about 50 mg / mL, from about 35 to about 45 mg / mL, eg, about 33. It is in the range of ~ about 48 mg / mL sorbitol.

In certain embodiments, sucrose has a concentration of about 70-about 90 mg / mL. In certain embodiments, the composition comprises from about 60 to about 100 mg / mL sucrose, from about 65 to about 95 mg / mL, from about 70 to about 90 mg / mL, from about 75 to about 85 mg / mL, eg, It ranges from about 72 to about 84 mg / mL sucrose.

In certain embodiments, the polyol is mannitol. In certain embodiments, the composition comprises about 10 to about 100 mg / mL, or about 20 to about 80, about 20 to about 70, about 30 to about 60, about 30 to about 50 mg / mL of mannitol, eg. , About 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 mg / mL.

In certain embodiments, the lyophilized formulation of the present disclosure comprises AS-DVD-Ig, a buffer having a molar concentration of about 5 to about 50 mM, and a polyol, which formulation is about 4.5 to about 7. It has a pH of .5.

In addition to the buffer, surfactants may be added to the lyophilized formulation, for example for additional stability. Examples of surfactants include nonionic detergents such as polysorbate (eg, polysorbate 20, 60, 80) or poloxamer (eg, poloxamer 188). In certain embodiments, the amount of detergent added reduces the aggregation of the formulated LS-DVD-Ig protein and / or minimizes the formation of microparticles in the formulation and / or The amount is such that adsorption is reduced.

In certain embodiments, the lyophilized formulation contains detergent polysorbate 80 or Tween 80. Tween 80 is a term used to describe polyoxyethylene (20) sorbitan monooleate. In certain embodiments, the formulation comprises about 0.001 to about 0.1% polysorbate 80, or about 0.005 and about 0.05%, 20 polysorbate 80, such as about 0.001, about 0. It contains .005, about 0.01, about 0.05, or about 0.1% polysorbate 80. In certain embodiments, about 0.01% polysorbate 80 is found in the formulations of the present disclosure.

In certain embodiments, the lyophilized formulation of the present disclosure comprises LS-DVD-Ig, a buffer having a molar concentration of about 5 to about 50 mM, and a surfactant, which formulation is from about 4.5 to. It has a pH of about 7.5. In certain embodiments, the surfactant is polysorbate, such as polysorbate 80 or polysorbate 20. In certain embodiments, the polysorbate has a concentration of about 0.005% to about 0.02%.

In certain embodiments, the lyophilized formulation of the present disclosure comprises LS-DVD-Ig, a buffer solution having a molar concentration of about 5 to about 50 mM, a surfactant, and a polyol, which formulation is about 4. It has a pH of 5 to about 7.5. In certain embodiments, the formulation comprises LS-DVD-Ig, buffer (eg, histidine), polysorbate (eg, polysorbate 80), and sugar alcohol (eg, mannitol or sorbitol). In certain embodiments, the formulation comprises LS-DVD-Ig, buffer (eg, histidine), polysorbate, such as polysorbate 80, and non-reducing sugar, such as sucrose.

The lyophilized formulations described herein are first made as "pre-lyophilized", which pre-dried formulations are pre-lyophilized formulations. The amount of protein present in the pre-dried preparation is determined in consideration of the desired dose volume, administration mode (s), and the like. For example, the starting concentration of LS-DVD-Ig protein can be from about 2 mg / mL to about 50 mg / mL.

In certain embodiments, the DVD-Ig proteins used in the lyophilized formulations of the present disclosure specifically bind to TNF / IL-17 and are heavy and light chains set forth in SEQ ID NOs: 62 and 63. Contains the amino acid sequence corresponding to the CDR. In certain embodiments, the anti-TNF / IL-17-Ig protein comprises the amino acid sequences corresponding to the heavy and light chain variable regions set forth in SEQ ID NOs: 62 and 63. In certain embodiments, the anti-TNF / IL-17-Ig protein comprises the amino acid sequences corresponding to the heavy and light chains set forth in SEQ ID NOs: 62 and 63.

In certain embodiments, the lyophilized formulations of the present disclosure have anti-TNF / IL-17 DVD-Ig proteins (eg, amino acid sequences corresponding to the heavy and light chain CDRs set forth in SEQ ID NOs: 62 and 63). An anti-TNF / IL-17 DVD-Ig protein comprising a heavy chain and a light chain comprising, or a heavy chain and a light chain comprising an amino acid sequence corresponding to the heavy and light chain variable regions set forth in SEQ ID NOs: 62 and 63. Contains anti-TNF / IL-17 DVD-Ig protein, or DVD-A), histidine or acetate buffer, and sucrose or sorbitol, the formulation having a pH of about 5 to about 6 and a concentration of this protein. Is over about 50 mg / mL when reconstituted.

In certain embodiments, the DVD-Ig protein used in the lyophilized formulations of the present disclosure specifically binds IL1α / IL-1β and comprises the amino acid sequences set forth in SEQ ID NOs: 66 and 67, respectively. Includes heavy and light chain sequences. In certain embodiments, the DVD-Ig protein used in the lyophilized formulation specifically binds IL-1α / IL-1β and is set forth in SEQ ID NOs: 66 and 67 for heavy and light chain CDRs. Contains the amino acid sequence corresponding to. In certain embodiments, the anti-IL-1α / IL-1β-Ig protein comprises the amino acid sequences corresponding to the heavy and light chain variable regions set forth in SEQ ID NOs: 66 and 67. In certain embodiments, the anti-IL-1α / IL-1β-Ig protein comprises the amino acid sequences corresponding to the heavy and light chains set forth in SEQ ID NOs: 66 and 67.

In certain embodiments, the lyophilized formulations of the present disclosure are heavy chains comprising anti-IL1α / IL1β DVD-Ig proteins (eg, amino acid sequences corresponding to the heavy and light chain CDRs set forth in SEQ ID NOs: 66 and 67). Anti-IL1α / IL1β DVD-Ig protein, including and light chain, or anti-TNF / containing heavy and light chains comprising the amino acid sequences corresponding to the heavy and light chain variable regions set forth in SEQ ID NOs: 66 and 67. Includes IL-17 DVD-Ig protein, or DVD-C), histidine buffer, and sucrose or sorbitol, the formulation has a pH of about 5 to about 6, and this protein concentration is reconstituted. It is over about 50 mg / mL. In certain embodiments, the aqueous formulation has a pH in the range of about 5 to about 5.5, eg, about 5.4.

In certain embodiments, the DVD-Ig protein used in the lyophilized formulations of the present disclosure specifically binds to TNF / PGE2 to the heavy and light chain CDRs set forth in SEQ ID NOs: 64 and 65. Contains the corresponding amino acid sequence. In certain embodiments, the anti-TNF / PGE2-Ig protein comprises the amino acid sequences corresponding to the heavy and light chain variable regions set forth in SEQ ID NOs: 64 and 65. In certain embodiments, the anti-TNF / PGE2-Ig protein comprises the amino acid sequences corresponding to the heavy and light chains set forth in SEQ ID NOs: 64 and 65.

In certain embodiments, the lyophilized formulations of the present disclosure comprise a DVD-Ig protein that specifically binds to PLL4 / VEGF and are heavy and light as set forth in SEQ ID NOs: 28 and 29 or SEQ ID NOs: 72 and 73. Contains the amino acid sequence corresponding to the chain CDR. In certain embodiments, the anti-DLL4 / VEGF-Ig protein comprises the amino acid sequences corresponding to the heavy and light chain variable regions set forth in SEQ ID NOs: 28 and 29 or SEQ ID NOs: 72 and 73. In certain embodiments, the anti-DLL4 / VEGF-Ig protein comprises the amino acid sequences corresponding to the heavy and light chains set forth in SEQ ID NOs: 28 and 29 or SEQ ID NOs: 72 and 73.

In certain embodiments, the lyophilized formulations of the present disclosure comprise a DVD-Ig protein that specifically binds IL12 / IL18 and is an amino acid corresponding to the heavy and light chain CDRs set forth in SEQ ID NOs: 70 and 71. Contains an array. In certain embodiments, the anti-IL12 / IL18-Ig protein comprises the amino acid sequences corresponding to the heavy and light chain variable regions set forth in SEQ ID NOs: 70 and 71. In certain embodiments, the anti-IL12 / IL18-Ig protein comprises the amino acid sequences corresponding to the heavy and light chains set forth in SEQ ID NOs: 70 and 71.

Freeze-drying may be carried out according to methods known in the art. Hull50 (TM) (Hull, USA) or GT20. TM. Many different lyophilizers are available for this purpose, such as (Leybold-Heraeus, Germany) lyophilizers. Freeze-drying is carried out by freezing the formulation and then sublimating the ice from the frozen contents at a temperature suitable for primary drying. Under this condition, the product temperature is below the co-melting point or decay temperature of the formulation. Typically, the shelf temperature for primary drying will be in the range of about -30 to 25 ° C., with suitable pressures, typically in the range of about 50-250 mTorr (provided that the product is primary). It must remain frozen during drying). The size and type of formulation, container (eg, glass vial) holding the sample, and the volume of the liquid will primarily define the time required for drying, which can range from hours to days. It can be in the range (eg 40-60 hours). Optionally, a secondary drying step may also be performed depending on the moisture level of the desired residue in the product. The temperature at which the secondary drying takes place is mainly in the range of about 0-40 ° C., depending on the type and size of the container used and the type of protein. For example, the shelf temperature over the entire freeze-drying water removal step may be about 15-30 ° C (eg, about 20 ° C). The time and pressure required for secondary drying will, for example, produce a suitable lyophilized cake, depending on temperature and other parameters. The secondary drying time is defined by the desired residual moisture level in the product and typically takes at least about 5 hours (eg 10-15 hours). The pressure may be the same as the pressure used during the drying process. The lyophilization conditions can be varied depending on the formulation and vial size.

Prior to administration to the patient, the lyophilized formulation was reconstituted with a pharmaceutically acceptable diluent so that the protein concentration in the reconstituted formulation was at least about 2 mg / mL, eg, about 2 mg / mL. It is from mL to about 100 mg / mL, as an alternative from about 10 mg / mL to about 90 mg / mL, and as an alternative from about 30 mg / mL to about 50 mg / mL. Such high protein concentrations in the reconstituted formulation are considered to be particularly useful when subcutaneous delivery of the reconstituted formulation is intended. However, for other routes of administration, such as intravenous administration, lower concentrations of protein in the reconstituted formulation may be desired (eg, about 2-50 mg / mL, or about 3 in the reconstituted formulation). ~ 40 mg / mL protein). In certain embodiments, the protein concentration in the reconstituted formulation is significantly higher than the protein concentration in the pre-dried formulation. Reconstitution generally occurs at a temperature of about 25 degrees Celsius to ensure complete hydration, but other temperatures may be used as desired. The time required for reconstruction will depend, for example, on the type of diluent, excipients (s) and amount of protein. Examples of diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffered solution (eg, phosphate buffered saline), sterile saline, Ringer's solution or dextrose solution. Diluents are optional and contain preservatives. Examples of preservatives have been described above, of which aromatic alcohols such as benzyl or phenolic alcohol are preferred preservatives. The amount of preservative used is determined by assessing different preservative concentrations for protein compatibility and preservative efficacy testing. For example, if the preservative is an aromatic alcohol (such as benzyl alcohol), it may be present in an amount of about 0.1-2.0% and preferably about 0.5-1.5%, but most preferably. Can be present in an amount of about 1.0-1.2%.

V. Uses of the Disclosure The formulations of the present disclosure may be used therapeutically, i.e., both in vivo and as reagents for in vitro or insight purposes. The methods of the present disclosure may also be used to make aqueous formulations with properties that are advantageous for therapeutic use. The aqueous formulation may be used as a pharmaceutical formulation to treat the disorder in the subject.

The formulations of the present disclosure may be used to treat any disorder for which this therapeutic protein is therapeutically suitable. A "disorder" is any condition that may benefit from treatment with this protein. This includes chronic and acute disorders or illnesses, including pathological conditions that make mammals vulnerable to the disorder in question. In the case of anti-TNF DVD-Ig, the therapeutically effective amount of DVD-Ig protein is autoimmune diseases such as rheumatoid arthritis, intestinal disorders such as Crohn's disease, spondyloarthropathies such as ankylosing spondylitis, and skin disorders such as psoriasis. May be administered to treat. In the case of anti-IL-12 DVD-Ig, a therapeutically effective amount of DVD-Ig protein may be administered to treat neuropathy such as multiple sclerosis or skin disorders such as psoriasis. Other examples of disorders that may be treated with the formulations of the present disclosure include cancer, including breast cancer, leukemia, lymphoma, and colorectal cancer.

The term "subject" is intended to include living organisms such as prokaryotes and eukaryotes. Examples of subjects include mammals such as humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In a specific embodiment of the present disclosure, the subject is a human.

The term "treatment" refers to both therapeutic and prophylactic or proactive measures. Those in need of treatment include those who already have a disability and those whose disability should be prevented.

Aqueous formulations may be administered to mammals in need of treatment, including humans, according to known methods of administration. Examples of administration methods include parenteral, subcutaneous, intramuscular, intravenous, intra-articular, intra-bronchial, intra-abdominal, intracapsular, intrachondral, intracavitary, intracavitary, intracerebral, intraventricular, Intracolon, cervical, gastric, liver, myocardium, bone, pelvis, peritoneum, abdominal cavity, thoracic cavity, prostate, lung, rectum, kidney, retina, spine, Intrathecal, intrathoracic, intrauterine, intravesical, bolus, vagina, rectum, buccal, sublingual, intranasal, and percutaneous.

Appropriate dosages of protein (“therapeutically effective amounts”) are, for example, the condition of the subject to be treated, the severity and process of the condition, whether the protein is administered for prophylactic or therapeutic purposes, previous treatments, the patient's. It will depend on the clinical history and response to the protein, the type of protein used, and the discretion of the attending physician. The protein may be administered to the patient at one time or over a series of treatments and may be administered to the patient at any time after diagnosis. The protein may be administered as the sole treatment or in combination with other drugs or treatments that are useful in treating the condition in question.

The actual dosage level of the AS-DVD-Ig protein or LS-DVD-Ig protein or active ingredient in the pharmaceutical formulation of the present disclosure is not toxic to the patient and is specific to the patient, composition, and mode of administration. In contrast, it may be modified to obtain an amount of active ingredient that is effective in achieving the desired therapeutic response.

The dosage level selected is the activity of the AS-DVD-Ig protein or LS-DVD-Ig protein found in the formulation, the route of administration, the duration of administration, the rate of excretion of the particular compound used, the duration of treatment. , Other drugs, compounds, and / or materials used in combination with the particular compound used, age, gender, weight, pathology, general health, and previous medical history, and medical technology of the patient being treated. Will depend on a variety of factors, including similar factors well known in.

In certain embodiments of the present disclosure, the dosage of AS-DVD-Ig protein in the formulation is from about 1 to about 250 mg. In certain embodiments, the dosage of AS-DVD-Ig protein in the formulation is about 30-about 140 mg, about 40-about 120 mg, about 50-about 110 mg, about 60-about 100 mg, or about 70-about. It is 90 mg. In a further embodiment, the composition comprises about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190. , 200, 210, 220, 230, 240, or 250 mg of AS-DVD-Ig protein dosage.

In certain embodiments of the present disclosure, the dosage of LS-DVD-Ig protein in the formulation (at reconstitution) is from about 1 to about 250 mg. In a further embodiment, the dosage of LS-DVD-Ig in the formulation is about 30-about 140 mg, about 40-about 120 mg, about 50-about 110 mg, about 60-about 100 mg, or about 70-about 90 mg. .. In a further embodiment, the composition comprises about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190. , 200, 210, 220, 230, 240, or 250 mg of LS-DVD-Ig dosage.

The formulations of the present disclosure overcome common problems known in formulation development, including problems with protein aggregation, often associated with high concentrations of proteins, especially complex proteins such as DVD-Ig proteins. Therefore, in certain embodiments, the formulations of the present disclosure provide new means by which high levels of this new therapeutic protein form can be administered to a patient.

The examples presented herein describe formulations containing a bivariable domain immunoglobulin (DVD-Ig) protein with unexpected stability properties. Experiments have shown that certain DVD-Ig proteins, referred to as aqueous stable DVD-Ig (AS-DVD-Ig) proteins or freeze-dried stable DVD-Ig (LS-DVD-Ig) proteins, respectively. It was surprising in that it was stable in aqueous or lyophilized formulations, while other DVD-Ig proteins exhibited aggregation and instability when similarly formulated. The experiments show, for example, methods for identifying AS-DVD-Ig and LS-DVD-Ig proteins, as well as stable formulations thereof.

Materials and Methods The methods described herein were used in experiments performed to assess and monitor the stability of DVD-Ig proteins in aqueous and lyophilized formulations.

General Methods DVD-Ig protein formulations are subjected to general quality parameters (eg, pH), physical stability parameters (eg, clarity, color, particle contamination, and purity), and chemical stability parameters (eg, for example). , Deamidation, oxidation, and general chemical stability). Examples of tests include testing for visible particulate contamination, testing for the number of light blocking particles for invisible particles, and size exclusion high performance liquid chromatography (size exclusion chromatography (SEC) herein). Tests for purity such as (also referred to as) and ion exchange chromatography (IEC) were included.

Fine particle contamination (eg, visible particles) was determined by visual inspection. Invisible particles were monitored by a light blocking assay according to the United States Pharmacopeia (USP). The physicochemical stability of the formulation was assessed by the SEC, which allows detection of fragments and aggregates. SEC (for detection of fragments and hydrolysis) and IEC were performed to monitor chemical stability.

DVD-Ig Proteins Tested The DVD-Ig proteins tested in the examples provided herein are listed in Table 1. The sequences of the DVD-Ig proteins listed in Table 1 are provided in Table 65.

The DVD-Ig protein as a starting material was obtained after purification, which was over 95% monomeric.

Cation Exchange HPLC Method Cation exchange HPLC, a form of IEC, was used to determine the identity and purity of DVD-Ig protein formulations. The assay was performed with the parameters detailed below.

A Dionex ProPac (R) WCX-10 analytical column (Dionex Corp., Sunnyvale, CA) combined with a Dionex WCX-10G guard column (Dionex Corp., Sunnyvale, CA), limiting the upper limit column pressure to 210 bar. Executed. Mobile phase A consisted of 10 mM Na ₂ HPO ₄ (pH 6.0). This buffer is prepared by dissolving 4.97 g of anhydrous disodium hydrogen phosphate in approximately 3300 mL Milli-Q water, adjusting the pH to 7.0 using 1 M phosphoric acid, and adjusting the buffer volume to Milli. It was made by increasing the pH to 3500 mL with water and filtering the solution through a membrane filter. Mobile phase B consisted of 10 mM Na ₂ HPO ₄ and 500 mM NaCl (pH 6.0). This buffer is prepared by dissolving 2.56 g of disodium hydrogen phosphate in approximately 1500 mL of Milli-Q water, adjusting the pH to 6.0 using 1 M of phosphoric acid, and adjusting the buffer volume to Milli. It was made by increasing to 1800 mL with −Q water and filtering the solution through a membrane filter. A summary of the cation exchange HPLC method is given in Table 2.

For IL12 IL18 and DVD 66 DVD-Ig proteins, the mobile phase used was changed as follows:
Mobile phase A: 10 mM MES (pH 5.6), and mobile phase B: 10 mM MES + 500 mM NaCl (pH 5.6).

For IL1αILβ, the mobile phase used was changed as follows:
Mobile phase A: 20 mM phosphate (pH 8.0), and mobile phase B: 20 mM phosphate + 500 mM NaCl (pH 8.0).

Similar versions of IEC were used for various other DVD-Ig proteins.

Size Exclusion HPLC Method Size exclusion HPLC was used to determine the purity of the DVD-Ig protein solution. The assay was performed as outlined below.

TSK Gel Guard (VWR Scientific, Bridgeport, NJ, Catalog Number 08543, 6.0 mm x 4.0 cm, 7 μm), TSK Gel G3000SW (VWR Scientific, Bridgeport, NJ, Catalog Number 08541, 7.8 mm x 30) Combined, it was performed with an upper limit column pressure of 70 bar. The mobile phase consisted of _{100mM Na 2 HPO 4 / 200mM Na} 2 SO 4 (pH7.0). This buffer dissolves 49.68 g of anhydrous disodium hydrogen phosphate and 99.44 g of anhydrous sodium sulfate in approximately 3300 mL of Milli-Q water and adjusts the pH to 7.0 using 1 M of phosphoric acid. It was prepared by adjusting and increasing the buffer volume to 3500 mL with Milli-Q water and filtering the solution through a membrane filter.

The experimental parameters are listed as follows:
Flow rate: 0.3 mL / min,
Injection volume (comparable to 20 μg sample): 20 μL,
Column temperature: room temperature,
Autosampler temperature: 2-8 ° C,
Execution time: 50 minutes,
Elution: Uniform concentration gradient.

Detection was performed using a diode array detector using 214 nm wavelength (peak width over 0.1 minutes and 8 nm bandwidth) and 360 nm reference wavelength (100 nm bandwidth).

The test samples were injected in two series. Purity was determined by comparing the area of the DVD-Ig protein peak to the total area of all 214 nm absorption components in the sample, except for buffer-related peaks. High molecular weight aggregates and antibody fragments were degraded from intact DVD-Ig protein using this method.

Freeze / Thaw Assay A stable solution of DVD-Ig protein was measured using a repeated freeze / thaw assay. The DVD-Ig protein was frozen at −80 ° C. and then thawed at 30 ° C. in a water bath, and the resulting solution was analyzed by SEC for aggregation and / or by photoblocking assay for invisible particle formation. ..

Accelerated and real-time storage stability studies The pH and storage temperature of formulations are two important factors that affect protein stability during long-term storage of protein liquid and lyophilized formulations. To quickly gain insight into the success or failure of the formulation for long-term storage at lower temperatures (eg, 2-8 ° C) to mimic long-term storage, heat the protein formulation, eg, short-term storage at 40 ° C (acceleration). The effects of these factors were assessed by exposure to storage). Samples were also maintained at 2-8 ° C. to assess real-time storage stability.

Light-blocking assay A light-blocking assay was performed to measure the content of insoluble particulates in the agglutinated DVD-Ig protein solution. Measured by attaching a laminar air hood (Thermo Electron Corp., Asheville, NC, model number ULT2586-9-A40) to a light blocking measuring device (particle counter, model syringe, Klotz Bad Liebenzell, Germany, series S20037). Minimized foreign particle contamination inside. The light blocking analysis was performed as follows. A 3.5 mL sample was placed in a 5 mL round bottom tube under laminar air flow conditions. After the first 0.8 mL rinse, measurements were taken in n = 3 modes (0.8 mL per measurement) according to the manufacturer's specifications.

Differential scanning calorimetry (DSC)
Prior to DSC analysis, the DVD-Ig protein was dialyzed into a suitable buffer system using Slide-A-Lyzer Cassettes (Thermo Fisher Scientific, Waltham, MA). This buffer system (eg, 5 mM phosphate / 5 mM citrate was also used as a reference / blank for DSC measurements. Antibodies were analyzed at 1-2 mg / mL. Automatic VP-capillary DSC system ( MicroCal, Piscataway, NJ) was used. Molecular unfolding was studied by applying a scanning rate of 1 ° C./min over a temperature range of 25 ° C. to 95 ° C. Other measurement parameters were: T: Fitting period: 16 seconds; Pre-scanning wait time: 10 minutes; Feedback mode: None.

Gas-liquid interfacial vibration studies Vibration studies were performed in 6R glass vials on an HS 260 IKA shaker (Wilmington, NC) at a concentration of 1 mg / mL at a rate of 150 rpm (rpm). The optical densities of the samples were evaluated after various periods of vibration. Similarly, SEC was also performed on samples taken at various time points.

PEG Solubility PEG 3000 was used for solubility studies. A 50 w / v% solution of PEG 3000 was prepared in water. A small amount of aliquot of this solution was added to the protein stock solution in buffer at a concentration of 0.5 mg / mL. Note the total volume required at the beginning of the first signs of precipitation.

Authentic solubility To assess authentic solubility, DVD-Ig protein was concentrated and stored overnight at 5 ° C. The solution was visually inspected for precipitation, phase separation, turbidity, etc. The supernatant (or both phases) was confirmed for the dissolved concentration.

Near UV-CD
Near UV-CD scans were taken at 250-320 nm by a Jasco spectrometer (JASCO Analytical Instruments, Easton, MD) using 2 mL vials filled at a concentration of 1 mg / mL. The scanning speed was 50 nm / min, and an average of 3 scans were taken. Data were acquired after equilibrating the spectrometer by turning the lamp.

ATR-FTIR Spectroscopy FTIR scans were taken by Bruker ATR-FTIR (Bruker Optics, Billerica, MA) using a 10 μL solution at a concentration of 1 mg / mL. Scans were collected at 400-4000 cm- ¹ , the area was normalized to obtain a quadratic derivative, and then curve fitting was performed using Origin software (OriginLab, Northampton, MA).

Light Scattering Light scattering studies were performed by a Malvern Instruments Ltd., Worcestershire, UK, using a backscatter angle of 173 °. Toluene was used as the standard and buffers (eg, acetate, histidine, and Tris) were used as blanks. I used the automatic mode.

Dynamic scanning fluorescence quantification (DSF)
DSF was used to assess the propensity for protein unfolding. This technique involved adding the dye Cyclo Orange to a protein sample. This fluorescent dye is sensitive to hydrophobic surfaces and exhibits increased fluorescence in such an environment. Samples containing the dye were then heated and the fluorescence signal as a function of temperature was monitored. As the temperature increased, the protein began to unfold, exposing its hydrophobic inner surface. This led to dye binding to this region and a larger fluorescent signal. The temperature at which the signal begins to increase is the starting temperature ( _Ton ). Proteins with lower intrinsic stability are more prone to unfolding and have lower _Ton values than proteins with higher intrinsic stability. DSF also provides a high-treatment tool for rapid screening of clones in 96-well format, eliminating the potential limitation of larger samples and longer run times in DSC. 6 μL of 0.4X SYPRO Orange dye (Invitrogen, Carlsbad, CA) was added to 27 μL of DVD-Ig protein solution. The scanning speed was 1 ° C./min and the scans were taken at 25-75 ° C.

Freeze-drying method The DVD-Ig protein was freeze-dried according to a standard method, and the stability of the resulting freeze-dried product was investigated.

Sample preparation and lyophilization vials were plugged with autoclaved and dried lysing stoppers. The vial was then lyophilized in a lyophilizer. Typical cycles are shown below in Table 4. The sample was reconstituted into a 100 mg / mL DVD-Ig protein solution.

I. DVD-IG Protein Stability In Examples 1-3, the stability of the DVD-Ig protein is lower than that of the antibody due to the increased structural complexity of the DVD-Ig protein, eg, more easily aggregated. And demonstrate that it has a lower melting temperature.

Example 1. Thermodynamic Comparison of DVD-Ig Proteins and Antibodies Experiments were performed to determine the stability of DVD-Ig proteins compared to antibodies. Differential scanning calorimetry (DSC) of one IgG molecule (monoclonal antibody, mAb) and DVD-Ig was performed to determine the difference in thermodynamic properties between the two molecules. Specifically, DSC was performed to compare the thermodynamic properties of the exemplary antibody (briaquinumab, anti-IL12 monoclonal antibody) with that of the DVD-Ig protein (TNF / PGE2, DVD-B). Formulation information and DSC conditions are provided below in Example 2. Comparison of the DSC profile of briaquinumab with that of TNF / PGE2 (DVD-B) shows the difference in 3 domain unfolding vs. 4 domain unfolding (Fig. 1). It is also clear from FIG. 1 that the thermal unfolding of the DVD-Ig protein started earlier than that of the antibody, which is the overall thermodynamic stability (or intrinsic) of the DVD-Ig molecule. Stability) is lower than that of the antibody. While the DVD-Ig protein in FIG. 1 (DVD-B) had a DSC profile showing initial unfolding at about 50 ° C., this same DVD-Ig protein considered the results from further stability tests. It should be noted that it was characterized as an LS-DVD-Ig protein.

The thermodynamic stability of mAbs (eg, the initiation of unfolding as determined by dynamic scanning calorimetry) is generally greater than approximately 5 ° C. compared to the DVD-Ig protein panels listed in Table 1. There is generally no direct correlation between the unfolding temperature of the DVD-Ig protein group and the aggregation tendency, but using these values as a screen, the stability of a given group of DVD-Ig proteins Can be rated. Therefore, the starting temperature of unfolding can be used to identify the AS-DVD-Ig protein or the LS-DVD-Ig protein. As described in FIG. 4, the overall distribution indicates that the DVD-Ig protein generally has a lower unfolding temperature compared to the antibody. FIG. 4 provides results summarized from 16 different DVD-Ig proteins and 14 different antibodies. The assays performed in the studies described in FIG. 4 are similar to those described above and those in FIG.

Example 2. Effect of pH on the stability of DVD-Ig protein in solution The thermodynamic stability (intrinsic stability) of DVD-Ig protein formulated in a solution having a pH of 4, 6, or 8 , Differential scanning calorimetry (DSC) was used for evaluation. All formulations had a DVD-Ig protein concentration of 1 mg / mL in 5 mM citrate / 5 mM phosphate buffer. Heating was performed at a scanning rate of 1 ° C./min. The results showing the effect of pH on the stability of multiple DVD-Ig proteins are provided in Table 5 below. Tm1 to Tm4 described in Table 5 represent thermal melting / unfolding temperatures of various domains such as CH1, CH2, and CH3 domains. The stability of the two antibodies (adalimumab and briaquinumab) is also described for comparison.

As shown in Table 5, DVD-Ig proteins generally have an unfolding start temperature above about 50 ° C., and the melting temperature is therefore slightly lower than the melting temperature of antibodies and other stable proteins. Example 2 shows that the starting temperature for briaquinumab and adalimumab is around 60 ° C. at pH 6, while the average for DVD-Ig protein is around 53 ° C.

The data also show that the melting temperature of the DVD-Ig protein is higher at pH 6 and pH 8 than at pH 4. Therefore, pH affects the physicochemical stability of the DVD-Ig protein, which appears to be best at approximately pH 6.

To further assess the effect of solution pH on the stability of DVD-Ig protein preparations during long-term storage, DVD-Ig protein preparations are placed before storage (T0) or after 3 months of accelerated storage. (T3m) was analyzed using SEC. DVD in solution (1 mg / mL DVD-Ig protein in 5 mM citrate / 5 mM phosphate buffer in the presence of 80 mg / mL sucrose) formulated at a pH of 4, 6, or 8 -The storage stability of Ig protein was evaluated. For accelerated storage, samples were filled in sterile vials (approximately 500 μL each) and stored under controlled conditions (in a temperature chamber and in the absence of light) at 40 ° C. DVD-Ig protein monomer (Mon) aggregate (Agg), and fragment (Frag) percentages were determined using SEC and the results are presented in Table 6.

The results in Table 6 showed that the DVD-Ig protein could be initially formulated in the pH range of pH 4 to pH 8 and that the initial values of the freshly prepared sample at T0 did not show a significant difference. .. However, after accelerated storage, the DVD-Ig protein at pH 4 and pH 8 showed a significant loss of monomer and a corresponding increase in aggregates or fragmentation.

The stability of the DVD-Ig protein tested was highest around pH 6. All of the DVD-Ig proteins tested (including DVD 5, DVD 6, DVD 38, DVD 53, DVD 54, DVD 65, DVD 66, DVD 165, DVD 166, DVD 258, DVD 277, and DVD 278) Had a higher monomer percentage and a lower fragment percentage at pH 6 than at H4 or pH 8.

Nine of the twelve DVD-Ig proteins tested showed lower aggregate percentages at pH 6 than at pH 4, and for three DVD-Ig proteins showing the opposite pattern, pH 6 and pH 4 The difference in aggregate percentages in was very small (less than 2.7% difference). Also, 11 of the 12 DVD-Ig proteins tested showed lower aggregate percentages at pH 6 than at pH 8. Therefore, accelerated storage resulted in increased aggregate formation. However, the increase was lower than expected, especially at pH 6.

IL12 IL18 Effect of solution pH on storage stability of DVD-Ig protein formulation Having a solution pH of 4, 6, or 8 to assess the effect of solution pH on the stability of DVD-Ig protein formulation during storage. Before storing the DVD-Ig protein preparation (T0) or after storing it at 5 ° C., 40 ° C., or 50 ° C. for 4 days (4d), 7 days (7d), or 21 days (21d). Analyzed using SEC and IEC (see Tables 6, 7, and 8). The solution evaluated had an IL12-IL18 DVD-Ig concentration of 2 mg / mL and was in a buffer of 10 mM citrate and 10 mM phosphate. Samples were filled in sterile vials (approximately 500 μL each) and stored under controlled conditions (in a temperature chamber and in the absence of light). Percentages of DVD-Ig protein monomers, aggregates, and fragments were determined using SEC (see Table 7) and major species, acidic and basic species were rated using IEC. (See Table 8).

SEC data show that the IL12IL18 DVD-Ig protein formulation was the most stable at pH 6. At pH 6, stored IL12IL18 DVD-Ig protein formulations generally showed greater than 95% monomer and less than 2% aggregates. Even under accelerated storage conditions at 50 ° C. for 21 days, the formulation retained more than 90% of monomers and less than 5% of aggregates. The IL12IL18 DVD-Ig formulation was more stable at pH 6 than pH 4 and pH 8, especially under longer duration and higher temperature storage conditions (eg, 21 days, under 50 ° C. conditions). Based on these results, the IL12 / IL18 DVD-Ig protein would be considered an AS-DVD-Ig protein given the stability results after storage at 50 ° C.

IEC data for IL12IL18 DVD-Ig protein formulations at pH 4, 6, and 8 show that chemical degradation is pH and storage temperature dependent. The highest chemical stability was observed for samples stored at 5 ° C. Minor changes in stability detected after 21 days indicate that the IL12IL18 DVD-Ig protein is stable at this storage temperature, which is considered common in commercial biotherapeutic agents. It was. The highest chemical stability was observed for formulations at pH 6, although the difference in stability was small. The lack of chemical stability was even more pronounced at elevated storage temperatures of 40 ° C and 50 ° C.

Example 3. DVD-Ig protein aggregates more easily than monoclonal antibodies The effect of vibration on antibody-to-DVD-Ig protein aggregation was examined. Vibration is a stress that can lead to the aggregation of molecules. Sensitivity of DVD-Ig protein TNF / PGE2 (DVD-B) to post-vibration aggregation using a solution with a protein concentration of 1 mg / mL in a 6R vial (pH 6 solution, 10 mM citrate / 10 mM phosphorus). Phosphate) compared to the monoclonal antibody briaquinumab. A 6R vial is filled with a sample of 5 mL of protein solution and placed on an HS 260 IKA shaker (Wilmington, NC) at a rate of 150 rpm (rpm) over various lengths of time (0, 5, Vibrated for 24, 48, or 96 hours). Samples were identified for optical density at 500 nm, which provides a measurement of solution turbidity. Higher turbidity indicates greater aggregation and lower stability. The results are shown in Table 9 showing that the DVD-Ig protein aggregates more easily than the monoclonal antibody.

Vibration caused the DVD-Ig protein to form more invisible and visible aggregates than the monoclonal antibody, as indicated by the OD ₅₀₀ measurement, which indicates the turbidity of the solution. Therefore, after 48 hours of vibration, the DVD-Ig protein was more prone to colloidal instability and was therefore less stable than the monoclonal antibody. The higher visible aggregate formation of the DVD-Ig protein compared to the monoclonal antibody indicates that the DVD-Ig protein is generally less stable to shear stress in solution than the monoclonal antibody. These results also suggest that not all DVD-Ig proteins are as stable as monoclonal antibodies at pH 6.

II. Assay to identify stable aqueous DVD-IG protein (AS-DVD-IG)

Example 4. DVD-Ig proteins can be characterized as "aqueous stable" or "aqueous instability".
As discussed above, we found that formulations in which the DVD-Ig protein is in an aqueous state suffer certain problems, such as aggregation and / or fragmentation of the DVD-Ig protein monomer. I found it. The inventors conducted experiments and, surprisingly and unexpectedly, found that a subset of the DVD-Ig protein could be stably formulated in an aqueous state, even at high concentrations. The following example describes a SEC study, which surprisingly states that the DVD-Ig protein is aqueous stable, eg, the DVD-Ig protein has a monomeric relative (rel.) Peak area. It has been shown that it can be characterized as having a low percent variation or being aqueously unstable, eg, the DVD-Ig protein is either prone to aggregation and / or fragmentation. Remarkably, many of the DVD-Ig proteins tested were found to be aqueous or lyophilized. Due to the structural complexity of the DVD-Ig protein and the significant hydrophobic interactions at high concentrations, it was not expected that the DVD-Ig protein would be stable in high concentration formulations.

Accelerate protein liquid formulations by exposing various DVD-Ig proteins to short-term storage at elevated temperature to quickly gain insight into the success or failure of the formulation for long-term storage at lower temperatures (eg, 2-8 ° C.). The effect of storage temperature on protein stability during storage or long-term storage was assessed.

Stability Screening for 14 Days at High Concentration A DVD-Ig protein formulation with a concentration of 60 mg / mL was placed before storage (T = 0) or after 14 days of accelerated storage (T = 14 days). Analysis was performed using SEC (Table 10). The stability of stored DVD-Ig protein in solution (60 mg / mL 10 mM citrate / 10 mM phosphate buffer, including 80 mg / mL sucrose) was evaluated at 40 ° C. After the prescribed storage period, samples were removed and the effect of storage time on DVD-Ig protein stability was evaluated. Briefly, samples were filled in sterile vials (approximately 500 μL each) and stored under controlled conditions (in a temperature chamber and in the absence of light) at 40 ° C. At a given point in time, a sample of the prepared solution was taken out for analysis according to the sample removal scheme. DVD-Ig protein monomer (Mon) aggregate (Agg), and fragment (Frag) percentages were determined using SEC and the results are presented in Table 10.

Surprisingly, as shown in Table 10, the inventors found that the subset of DVD-Ig protein tested was stable when formulated in an aqueous state. Of the 12 DVD-Ig proteins tested, 10 (DVD 5, DVD 6, DVD 37, DVD 65, DVD 66, DVD 166, DVD 257, DVD 277, DVD 278, and DVD 282) were 14 After accelerating storage for days, it showed less than 26% aggregate formation and had more than 73% monomer. Five of the DVD-Ig proteins tested (DVD 6, DVD 65, DVD 66, DVD 166, and DVD 282) showed less than 10% aggregate formation and three of these (DVD 66). , DVD 166, and DVD 282) showed less than 5% aggregate formation.

As mentioned above, certain DVD-Ig proteins (“water-based stable DVD-Ig” proteins or “AS-DVD-Ig” proteins) are formulated in high concentrations (eg, concentrations above 60 mg / mL). Even when it is, it remains stable after 14 days of accelerated storage at 40 ° C. (eg, less than 6% monomer relative (rel.) Peak area change or less than 10%, as determined by SEC. Monomer relative (rel.) Peak area change). The majority of DVD-Ig proteins tended to aggregate during accelerated storage, as expected based on the general structure of DVD-Ig proteins and the stability studies described in Examples 1-3 (non-AS). -DVD-Ig protein). Thus, in certain embodiments, the cutoff for distinguishing the AS-DVD-Ig protein from the non-AS-DVD-Ig protein was stored at pH 6 above 50 mg / mL and at 40 ° C. for 14 days. Taken as less than 10% net aggregate formation, this is the non-limiting example above, where 5 of the 12 DVD-Ig proteins tested showed aggregate formation at this level. As seen in. In certain embodiments, a cutoff for distinguishing between AS-DVD-Ig protein and non-AS-DVD-Ig protein was 6 when stored at pH 6 above 50 mg / mL and at 40 ° C. for 14 days. Taken as less than or equal to the formation of net aggregates, which was taken in the non-limiting example above, where 4 of the 12 DVD-Ig proteins tested showed aggregate formation at this level. As you can see.

Many DVD-Ig proteins do not show low aggregation, eg, less than 1% aggregation after 21 days at 5 ° C. or less than 10% after storage at 40 ° C. for 21 days. For example, in an assay that examined monomer loss after 7 days in a solution with a TNF / IL13 DVD-Ig protein concentration of 50 mg / mL at either 4 ° C or 40 ° C, many DVD-Ig proteins were SEC. When determined by, showed an increase in monomeric loss. In some cases, the amount of monomer loss was negative due to increased monomer levels in these cases (eg, some of the agglomerates dissociated and the monomer was re-released. Formed, and thus the loss was apparently reduced). The third experiment tested TNF / SOST DVD-Ig protein in a solution with a DVD-Ig protein concentration of 50 mg / mL at 4 ° C. Many of the DVD-Ig proteins showed increased monomer loss (determined by SEC), as in the experiments on TNF / IL13 DVD-Ig proteins.

Notably, the above assays can also be used to identify lyophilized and stable DVD-immunoglobulin (LS-DVD-Ig) proteins. A cutoff for distinguishing between LS-DVD-Ig protein and non-LS-DVD-Ig protein was 15% or less of net aggregates stored at pH 6 above 50 mg / mL and at 40 ° C. for 14 days. Taken as a formation. Therefore, the DVD-Ig protein tested in the above assay that results in less than 10% or less than 6% aggregation, or less than 10% or less than 6% monomeric relative (rel.) Peak area variation is AS. DVD-Ig proteins that are considered both -DVD-Ig protein and LS-DVD-Ig protein and result in less than 15% aggregation or less than 15% monomeric relative (rel.) Peak area variation are LS-DVD. -Ig protein is considered. Both the AS-DVD-Ig protein and the LS-DVD-Ig protein correspond only to a certain percentage of the overall DVD-Ig protein tested.

III. Stability of Non-AS-DVD-IG Protein in Formulation The following example is a non-AS-DVD-Ig protein in formula compared to AS-DVD-Ig protein (described in Sections IV-VIII). Data are provided showing the stability (which is described in Example 4 above, which fails the aggregation test, i.e. shows, for example, greater than 6% aggregation).

Example 5: Effect of Concentration of Non-AS-DVD-Ig Protein on Storage Stability, 1, 2, 5, 10, 25, 50 to assess the effect of protein concentration on long-term storage stability , And an example non-AS-DVD-Ig protein formulation at a concentration of 75 mg / mL were subjected to storage at 40 ° C. for 14 days. The formulation was pH 6 and was in a buffer of 15 mM histidine alone. Samples were filled in sterile vials (approximately 500 μL each) and stored under controlled conditions (in a temperature chamber and in the absence of light). Samples were analyzed using SEC to determine the percentage of aggregates after storage. The resulting data are provided in Table 11.

The data in Table 11 show that under the conditions tested (ie, pH 6, 15 mM histidine buffer), DVD-B becomes unstable, i.e. high proportion, after storage at 40 ° C., which reached high concentrations, for 14 days. It is shown that agglomerates of. The percentage of aggregates formed exceeded 18% at concentrations above 25 mg / mL. Therefore, DVD-B, a non-AS-DVD-Ig protein, was not stable in histidine buffer, as evidenced by increased aggregation during storage.

Example 6: Effect of pH, Ionic Strength, and Concentration on Storage Stability of Example Non-AS-DVD-Ig Proteins pH, Ionic Strength, and Concentration on Storage Stability of DVD-Ig Protein in Solution Various formulations of DVD-B (5 mg / mL and 100 mg / mL) were evaluated at 40 ° C and 5 ° C to assess the effect of concentration. After the prescribed storage period, samples were removed and the effect of storage time on DVD-Ig protein stability was evaluated. The following buffers were used: acetate at pH 4.5, histidine at pH 6, and tris at pH 8. A buffer solution having a concentration of 2 mM was used for the 1 mM ionic strength solution, and a buffer solution having a concentration of 10 mM was used for the 20 and 100 mM ionic strength solutions (sodium chloride was used to further maintain the ionic strength). Samples were each filled in approximately 500 μL sterile vials and stored under controlled conditions in a temperature chamber and in the absence of light. After 3 months (5C, 3m) at 5 ° C. or 21 days (40C, 21d) at 40 ° C., samples of the prepared solution were analyzed using SEC. The number of net aggregates measured using SEC is shown in Table 12. Tables 13 and 14 further show that the addition of different stabilizers / excipients (eg, sucrose and Tween80) did not result in a significant improvement in the percentage of monomer remaining after the specified time point (the method described above). Used and obtained results). Negative values of net aggregate values at the first time point for low concentration samples also indicate the initial formation of reversible aggregates.

Addition of polyol to the formulation resulted in a slight improvement in the monomer content of DVD-B at 1 mg / mL and a decrease in condensate levels.

The addition of polyol to the formulation resulted in a slight improvement in the monomer content of DVD-B at 100 mg / mL and a decrease in condensate levels.

The data show that DVD-B was not very stable even at pH 6 although various solution conditions were analyzed (see, eg, Table 13). Also, at pH 6, ionic strength did not show a consistent relationship with net aggregate formation. Poor stability is exhibited by the formation of high agglomerates even under storage conditions of 5 ° C. In addition, the addition of polyols and / or surfactants also improved DVD-B aggregation (see Tables 13, 14, and 15).

As mentioned above in Examples 1-3, many DVD-Ig proteins are endogenously unstable. However, surprisingly, certain DVD-Ig proteins can be characterized as stable, as described in Example 4. The experiments described in the Examples below demonstrate that the AS-DVD-Ig protein can be unexpectedly and stably formulated even at high concentrations, despite differences in amino acid sequences. The examples below are in contrast to Examples 5 and 6 and show the unsuccessful formulation of non-AS-DVD-Ig protein.

IV. The AS-DVD-IG protein is stable in formulations containing buffers in the pH range of 4.5-7.5.

Example 7: Effect of Buffer Concentration on DVD-Ig Protein Stability The concentration of buffer, eg, histidine, is one of the key factors that can affect protein stability during accelerated / long-term storage of protein liquid formulations. There is one. This effect was assessed by exposing the protein to warming and short-term storage at real-time temperatures in order to quickly gain insight into stable formulations for long-term storage at lower temperatures (eg, 2-8 ° C.).

The storage stability of DVD-Ig protein in solution was evaluated at 40 ° C and 5 ° C. After the prescribed storage period, samples were removed and the effect of storage time on DVD-Ig protein stability was evaluated. Evaluated histidine concentrations include 0, 5, 15, 50, and 200 mM.

Samples were filled in sterile vials (approximately 500 μL each) and stored under controlled conditions (in a temperature chamber and in the absence of light). After 7 and 21 days, samples of the prepared solution were analyzed using SEC and IEC.

＊沈殿は、不溶性である可視的凝集体が観察されるかどうか（有または無）を示す。

* Precipitation indicates whether insoluble visible aggregates are observed (yes or no).

Tables 16 and 17 suggest that histidine concentrations in the range of about 5 to about 50 mM provided optimal stability, depending on the amount of monomer remaining at different time points and during the formation of insoluble aggregates. Show that. A concentration of about 200 mM histidine resulted in the formation of insoluble aggregates in some cases (see Precipitation Index in Table 16). The 0 mM histidine formulation showed increased aggregation, as indicated by the formation of insoluble and soluble aggregates in some cases (in some cases the soluble aggregate was 0 at a histidine concentration of 5 mM). It was higher than that of). Second, pH is expected to be well maintained over longer storage times in formulations containing histidine.

The error in the IEC readings is usually higher (2-3% variation) compared to the SEC readings for the same formulation. Thus, taking that into account, no significant differences were observed within the formulation when assayed by IEC, which resulted in chemical stability as the chemical stability was not significantly affected by the molar concentration of histidine. It is shown to be independent of buffer concentration (generally in contrast to aggregation tendency).

Example 8: Example of stability of AS-DVD Ig protein (DVD-C anti-IL1 alpha / beta DVD-Ig protein) in solution Anti-IL1α / βDVD-Ig protein (DVD-C) at different pH and different Both 100 mg / mL and 1 mg / mL in different buffers at temperature were rated for stability over time. The buffers tested on 100 mg / mL DVD-C were 15 mM acetate (pH 4), 15 mM acetate (pH 5), 15 mM histidine (pH 5.5), 15 mM succinate (pH 5.5), 15 mM histidine (pH 6). .0), water (without buffer) (pH 6.0), 15 mM citrate (pH 6.0), 15 mM histidine (pH 6.5), and 15 mM tris (pH 8.0). The buffer tested on 1 mg / mL DVD-C contained 10 mM citrate + 10 mM phosphate buffer at pH 3, 4, 5, 6, 7, or 8.

Samples were stored at 50 ° C, 40 ° C, 25 ° C, and 5 ° C. At a particular point in time, samples were taken and evaluated for stability. Physical stability was evaluated by size exclusion chromatography (SE-HPLC or SEC), including% aggregates,% monomers,% fragments, and total species recovered were quantified. Chemical stability was assessed by weak cation exchange chromatography (IEX-HPLC or IEC), including quantified% acidic species,% major species, and% basic species.

Tables 18 and 19 describe the stability of DVD-C at 100 mg / mL and 1 mg / mL, respectively, at various buffers and pH. Both size exclusion chromatography (SEC) data and ion exchange chromatography (IEC) data are shown in both Tables 17 and 18. Formulation and abbreviation solutions are given below each table.

製剤および省略形の解：
酢酸塩＝１５ｍＭ酢酸塩
ヒスチジン＝１５ｍＭヒスチジン
ｓｕｃｃ＝１５ｍＭコハク酸塩
水＝透析によって水中で製剤化
クエン酸塩＝１５ｍＭクエン酸塩
トリス＝１５ｍＭトリス
ＨＭＷ％＝ＳＥＣによって定量した高分子量種のパーセンテージ
Ｍ％＝ＳＥＣによって定量した単量体のパーセンテージ
ＬＭＷ％＝ＳＥＣによって定量した低分子量種（断片）のパーセンテージ
ＡＵＣ＝ＳＥＣ曲線の総積分面積
酸性％＝ＩＥＣによって定量した、主要種に対する酸性種のパーセンテージ
主要物質％＝ＩＥＣによって定量した主要種のパーセンテージ
塩基性％＝ＩＥＸによって定量した、主要種に対する塩基性種のパーセンテージ
Ｔ０＝時間ゼロ
Ｔ７ｄ＝７日間の貯蔵
Ｔ１ｍｏ＝１ヶ月間の貯蔵
Ｔ３ｍｏ＝３ヶ月間の貯蔵

Formulations and abbreviation solutions:
Acetate = 15 mM acetate histidine = 15 mM histidine base = 15 mM succinate Water = formulated in water by dialysis Citrate = 15 mM citrate Tris = 15 mM Tris HMW% = percentage of high molecular weight species quantified by SEC M% = Percentage of monomers quantified by SEC LMW% = Percentage of low molecular weight species (fragments) quantified by SEC AUC = Total integrated area of SEC curve Acid% = Percentage of acidic species relative to major species quantified by IEC Major substances % = Percentage of major species quantified by IEC Basic% = Percentage of basic species relative to major species quantified by IEX T0 = Zero time T7d = 7 days storage T1mo = 1 month storage T3mo = 3 months storage

省略形の解：
ＨＭＷ％＝ＳＥＣによって定量した高分子量種のパーセンテージ
Ｍ％＝ＳＥＣによって定量した単量体のパーセンテージ
ＬＭＷ％＝ＳＥＣによって定量した低分子量種（断片）のパーセンテージ
ＡＵＣ＝ＳＥＣ曲線の総積分面積
酸性％＝ＩＥＸによって定量した、主要種に対する酸性種のパーセンテージ
主要物質％＝ＩＥＸによって定量した主要種のパーセンテージ
塩基性％＝ＩＥＸによって定量した、主要種に対する塩基性種のパーセンテージ
ＮＤ＝種を検出せず
ＮＲ＝アッセイを実施せず
０＝時間ゼロ
７ｄ＝７日間の貯蔵
２１ｄ＝１ヶ月間の貯蔵
３ｍｏ＝３ヶ月間の貯蔵
６．５ｍｏ＝６．５ヶ月間の貯蔵

Abbreviated solution:
HMW% = Percentage of high molecular weight species quantified by SEC M% = Percentage of monomers quantified by SEC LMW% = Percentage of low molecular weight species (fragments) quantified by SEC AUC = Total integrated area of SEC curve Acid% = Percentage of acidic species to major species quantified by IEX Major substance% = Percentage of major species quantified by IEX Basic% = Percentage of basic species to major species quantified by IEX ND = No species detected NR = No assay performed 0 = zero time 7d = 7 days storage 21d = 1 month storage 3mo = 3 months storage 6.5mo = 6.5 months storage

The molecule was completely degraded during dialysis at pH 3. No species were detected by SEC or IEC after dialysis under this condition at zero time. Storage at pH 4 and 50 ° C. also produced the same results. Both results are indicated by ND.

In addition, the thermal stability of DVD-C was assessed by differential scanning calorimetry (DSC). Thermal stability was assessed with 1.0 mg / mL molecules formulated in 10 mM citrate + 10 mM phosphate buffer at pH 4, 5, 6, 7, or 8. Higher starting temperature unfolding, or higher domain midpoint temperature unfolding, means better thermal stability. Thermal stability at different pHes often correlates with the long-term stability of molecules formulated at these pHs. Therefore, DSC data can help identify the most stable and least stable pH of the molecule.

As mentioned above, the stability of DVD-C when stored at three different temperatures (40 ° C, 25 ° C, or 5 ° C) was tested at a number of different buffers and pH. The concentration of DVD-C ranged from 1 mg / mL to 100 mg / mL. At 100 mg / mL, the buffer contained acetate, histidine, succinate, citrate, and tris. DVD-C was also formulated in fresh water. The pH of the formulation ranged from 4 to 8. Proteins were formulated at 1 mg / mL in citrate-phosphate buffer at pH ranging from 3-8. Immediately after formulation, a sample of DVD-Ig protein was stored at the temperatures described above. At specific time points, aliquots were taken and evaluated for physical stability by SEC and chemical stability by weak cation exchange (WCX).

Overall, the data showed that DVD-C was stable outside of pH 3 and pH 4. Specifically, the data showed that the physical stability of DVD-C was best around pH 5.5 and the chemical stability was best around pH 6.0. Histidine and succinate have been determined to be the appropriate buffers for these pHs.

In addition, the thermal stability of DVD-C was assessed by differential scanning calorimetry (DSC) as shown in Table 18. Thermal stability was assessed on 1.0 mg / mL DVD-C formulated in citrate-phosphate buffer at pH 4-8. Higher start temperature unfolding (Ton) or higher domain midpoint temperature unfolding (Tm) means better thermal stability. Thermostability is likely to correlate with the long-term stability of DVD-Ig protein. The data show similar thermal stability in the pH range 5-8.

Example 9: Effect of polyol on stability of AS-DVD-Ig protein A tendency for protein unfolding to occur was assessed using dynamic scanning fluorescence (DSF). To assess the effect of these polyols on protein stability in solution, the effects of polyols such as sucrose and sorbitol were investigated. DVD-A, DVD-C, and IL12 / IL18 DVD-Ig proteins were used as exemplary AS-DVD-Ig proteins. As shown in Table 21 below, generally, AS-DVD-Ig proteins at various concentrations (1,100,150 mg / mL) include sucrose (eg, 40-160 mg / mL) and sorbitol (eg, 20). It is stable in the presence of ~ 80 mg / mL). Moreover, increased concentrations of sorbitol and sucrose provided resulted in slightly increased stability. Thus, the addition of saccharides to a buffer containing the protein formulation (eg, 15 mM histidine) slightly increases the stability of the AS-DVD-Ig protein.

As is apparent from the above data in Table 21, the polyols (eg, sorbitol and sucrose) are broad (20-160 mg / mL sucrose and 20-80 / mg / mL sorbitol) and AS-DVD in solution. -Ig protein stability can be improved. In addition, the data reveal a reduction in the initiation of unfolding due to increased DVD-Ig protein concentration, which in turn reduces thermodynamic stability. These data correlate with the observed instability, such as the aggregation observed with high concentration liquid formulations.

V. The AS-DVD-IG protein is stable in formulations containing buffers and polyols in the pH range 4.5-7.5.

Example 10: Effect of buffer on storage stability of DVD-A in formulations containing polyols

To assess the effect of the buffer on the storage stability of DVD-A in different buffers, the stability of the formulation was measured before storage (T0) or after 7 days of storage at 40 ° C. (accelerated storage). It was rated 7d) or 21 days later (21d). The AS-DVD-Ig protein DVD-A (85 mg / mL) is formulated in various buffers (15 mM citrate, 15 mM histidine, 15 mM arginine, 15 mM acetate, or water) at pH 5.2. did. Samples were filled in sterile vials (approximately 500 μL each) and stored under controlled conditions in a temperature chamber and in the absence of light. Samples are analyzed using SEC and the results are provided in Tables 22 and 23.

The SEC results provided in Table 22 show that the pH 5.2 histidine formulation had the lowest levels of aggregates compared to the pH 4 histidine formulation, but citrate and acetate buffers also had low levels of aggregates. Indicates that The citrate and acetate showed smaller amounts of soluble aggregates as measured by SEC, but the solution was visibly cloudy, indicating the formation of insoluble aggregates. However, considering the SEC measurements, the visible turbidity was not significant. Overall, citrate and acetate formulations were only slightly less stable than histidine formulations. Considering that the noise / error of IEC measurements is usually high, no significant difference in chemical stability was observed within the formulations presented in Table 23. The polyol alone formulation was also slightly less stable than the histidine formulation. Considering the overall stability properties of the protein identified as the AS-DVD-Ig protein, the slight differences observed in the SEC and IEC analyzes in Tables 22 and 23 are those of the buffer tested at pH 5.2. Each was shown to be stable. Therefore, the differences observed between the buffers tested at pH 5.2 use each to provide a stable formulation for the AS-DVD-Ig protein, including those in high concentrations. Shown to get.

VI. The AS-DVD-IG protein is stable in formulations containing buffers, polyols, and detergents in the pH range of 4.5-7.5.

Example 11: AS-DVD-Ig protein is stable in various histidine formulations containing surfactants and sugars.

The following examples affect the physicochemical stability of DVD-Ig protein in low and high concentration formulations during accelerated / real-time stability testing, pH, buffers, and excipients (surfactants and excipients). The effects of (including polyols) are described. To demonstrate that the DVD-Ig protein has significantly different protein properties compared to the monoclonal antibody, five different formulation conditions that are well known and used to maintain the stability of the monoclonal antibody are tested. did.

Storage stability of low (1 mg / mL) and higher (100 mg / mL) AS-DVD-Ig protein formulations using size exclusion chromatography (SEC) and ion exchange chromatography (IEC). Evaluation was performed according to three storage conditions: no storage (T0), 1 month (1 m, 5C) at a controlled temperature of 5 ° C., and 1 month (1 m, 40C) at a controlled temperature of 40 ° C. Formulations with a variety of pHs (choose a range of 5.25 to 7.2), buffers, and excipients were tested according to the following conditions:
1) pH 5.25 and pH 6, 15 mM histidine, 80 mg / mL sucrose, 0.01% Tween 80
2) pH 6, 15 mM histidine, 40 mg / mL sorbitol, 0.01% Tween 80
3) PBS at pH 6 and 7.2 (10 mM phosphate, 125 mM NaCl)
4) 20 mM glycine, 26 mg / mL glycerol, pH 6
5) Water, 0.01% Tween 80, pH 5 and 6

The results are presented in Tables 24-27 below. The data show that not all DVD-Ig proteins are stable at all tested pH and formulation conditions. DVD-B formed high amounts of aggregates under all solution conditions tested and was classified as a non-AS-DVD-Ig protein (according to the assay presented in Example 4). All other DVD-Ig proteins (preselected as AS-DVD-Ig proteins) responded normally and were stable.

The effects of these excipients were evaluated using sucrose, sorbitol, glycerol, and glycine. Tween 80 (polysorbate 80), a surfactant that provides stabilization against vibrational stress, was also used to assess its effect on the stability of high concentration solutions. The effect of salt concentration was evaluated by varying the ionic strength using sodium chloride.

In general, formulations at pH 6 or pH 5.2 in histidine buffer were effective against all AS-DVD-Ig proteins. Both sorbitol and sucrose each improved stability. Sucrose resulted in the formation of slightly less monomer after a defined time point. The presence of salt increases instability (less), especially at 1 mg / mL to a lesser extent, as shown in Table 24, IL12 / IL18 DVD-Ig protein at 100 mg / mL, and Table 27. Defined as a residue of monomer). The presence of glycerol and glycine resulted in less monomer residue after storage stability when compared to other formulations. For example, according to Table 24, the IL12IL18 DVD-Ig protein was slightly more stable at pH 6 in the presence of sucrose than sorbitol. The data also demonstrate that formulations from either low or very low ionic strength showed comparable stability over time.

SEC data assessing the rate of aggregate and / or fragment formation showed the physical stability of the DVD-Ig protein (see Tables 24 and 26). As shown in Table 26, all molecules did not show a large aggregation tendency at low concentrations. IEC data is an indicator of the chemical stability of the DVD-Ig protein. For example, deamidation results in the formation of acidic species (conversion of major species to acidic species). The generation of positively charged variants will lead to an increase in basic species. The formation of either of the two acidic or basic species exhibits instability as the formation of these two species results in an overall increase in% of the major species (see Tables 25 and 27). ..

The results in Tables 24-27 show that the AS-DVD-Ig protein is stable in formulations containing only detergents of pH 5-6, eg 0.01% Tween 80.

Example 12. Effects of Storage on the Stability of AS-DVD-Ig Protein (DVD-C) in Various Formulations The pH and storage temperature of protein formulations are two important factors affecting protein stability during accelerated / long-term storage. Is. The effects of these factors on exposing DVD-Ig protein to warming and short-term storage at real-time temperatures to gain insight into the success or failure of long-term storage formulations at lower temperatures (eg, 2-8 ° C.). Was rated.

The storage stability of DVD-C (100 mg / mL) in solution was evaluated in the formulation at 40 ° C. After the prescribed storage period, samples were removed and the effect of storage time on DVD-Ig protein stability was evaluated. Samples were filled in sterile vials (approximately 500 μL each) and stored under controlled conditions in a temperature chamber and in the absence of light. At a given point in time, a sample of the prepared solution was taken out for analysis according to the sample removal scheme. The resulting data are provided in Tables 28 and 29.

The data provided in Tables 28 and 29 showed that DVD-C was very stable (some instabilities) in that there was minimal loss at the monomer level during the test storage conditions. DVD-Ig protein, eg (compared to that in Example 5), will be classified as an AS-DVD-Ig protein.

Example 13: Effect of Surfactant on Stability of AS-DVD-Ig Protein in Formulations Containing Buffers and polyols Setting the formulation pH to more than 1 unit from the isoelectric point (pI) of the protein is possible. , Generally beneficial. The closer the formulation pH is to pI, the more generally the overall surface of the protein is considered uncharged, thus contributing to the protein-protein attraction of non-polar groups and therefore increasing non-covalent aggregation and instability. Let me. Vibration and agitation promote physical instability, which creates hydrophobic air / water interfaces, resulting in alignment of protein molecules at these interfaces and ultimately aggregation. Given that air is more hydrophobic than water, the interface between air and liquid is considered to be a modified surface where protein aggregation, especially with (partially) unfolding, can occur. The effective air-water interface can be increased by vibration or agitation.

In the next example, the effect of various concentrations of detergent (eg, Tween 80) on the instability of the example DVD-Ig protein was evaluated. Studies were conducted in the absence or presence of polyol sucrose. The data presented in Tables 30 and 31 compare different detergent concentrations (0-2 mg / mL) in histidine buffer at pH 5.2 or 5.4 with and without sucrose (80 mg / mL). To do. The results of the turbidity measurement showed that the surfactant (Tween 80) in the concentration range of 0.05 mg / mL to 2 mg / mL generally provided the AS-DVD-Ig protein with stability against shear / denaturing stress. Shown. Turbidity increased when the detergent concentration was reduced to 0.01 mg / mL. Similar observations were made for the AS-DVD-Ig protein in the presence of sucrose. All studies were performed on 15 mM histidine at a DVD-Ig protein concentration of 1 mg / mL. Corresponding SEC analysis showed that, in general, the highest loss in monomeric relative (rel.)% For samples containing 0.05-0.1 mg / mL Tween 80 yielded the most stable formulations. It is shown that the amount of Tween 80 obtained is 0.5 mg / mL to 2 mg / mL. Turbidity measurements at 500 nm using UV light are listed in Table 30, and SEC analysis of samples is listed in Table 31. The pH range was also tested in the formulations described below.

＊ＳＥＣデータは、表３０の０および９６ｈの振動試料に対応する。

* SEC data correspond to the vibration samples 0 and 96h in Table 30.

Example 14: Effect of Surfactant Concentration on Stability of IL12IL18 DVD-Ig Protein Measured in Vibration Study The following example was measured using optical densities of two different wavelengths, 350 and 500 nm. The effects of different concentrations (range of concentrations) of polysolvate 80 and poroxamer on the vibrational stability of IL12IL18 DVD-Ig protein (15 mM histidine + 80 mg / mL sculose) at a concentration of 1 mg / m at pH 6 are described. Samples were taken after 0, 24, 48, 120, and 240 hours.

As shown in Table 32, the addition of detergent increased the vibrational stability of the IL12 / IL18 DVD-Ig protein. Polysorbate concentrations in the range of 0.05-2 mg / mL were determined to be most effective for the stability of the IL12 / IL18 DVD-Ig protein, with poloxamers in the range of 0.1-2 w / v%. Determined to be the most effective. DVD-B was also tested and showed similar stability when tested in this particular vibration assay.

Example 15: Effect of polyol (sucrose and sorbitol) concentration on the stability of IL12IL18 and DVD-B DVD-Ig proteins The following examples are made by Avacta (York, UK), as described in the Method section. Effect of polyols ie sucrose and sorbitol in the presence of polysorbate 80 on the stability of IL12IL18 DVD-Ig protein measured at 3 mg / mL at pH 6 by endogenous fluorescence using the automatic high throughput device Optim-1000. Is shown. 9 μl of MCA was used in the study. Thermal scans were obtained using a scan rate of 1 ° C./min and scans were obtained from 25-75 ° C. All samples were freshly prepared from stock solutions for sucrose and sorbitol excipients.

Based on the data in Table 33, the stability of the DVD-B Ig protein increased with increasing concentration of either polyol (sucrose or sorbitol). They were investigated because 100 mg / mL for sucrose and 60 mg / mL for sorbitol were determined to be the approximate maximum concentrations that would achieve osmolality. Sucrose in the range of 60-100 mg / mL was determined to be effective, while sorbitol in the range of 20-60 mg / mL was effective for stability. In contrast, the results shown in Table 33 for the IL12 / IL18 DVD-Ig protein showed no improvement in stability when measured by DSF.

Example 16: Effect of pH and histidine concentration (at pH 6) on storage stability of DVD-B and IL12IL18 DVD-Ig proteins at 100 mg / mL The following example uses size exclusion chromatography (SEC). The effect of histidine concentration (range 0 to 200 mM) on the storage stability of DVD-B and IL12IL18 DVD-Ig proteins as measured in the above is shown. Samples were prepared and the storage stability of DVD-Ig protein in solution at 100 mg / mL was evaluated at 5 ° C and 40 ° C. After the prescribed storage period, samples were removed and the effect of storage time on DVD-Ig protein stability was evaluated. Briefly, samples were filled in sterile vials (approximately 500 μL each) and stored under controlled conditions (in a temperature chamber and in the absence of light) at 40 ° C. At a given point in time, a sample of the prepared solution was taken out for analysis according to the sample removal scheme.

Table 34 also affects the stability of the two DVD-Ig proteins in the pH range of 4.5-7.4 (pH 4.5 is 15 mM acetate, pH 6 is 15 mM histidine, and pH 7.4 is 15 mM phosphate). ) Is shown. The data show that the non-AS DVD-Ig protein DVD-B is unstable above pH 4.5, as shown by much higher levels of aggregate formation over time, while the AS-DVD-Ig protein It is shown that the stability of the IL1 IL18 DVD-Ig protein is maintained between a pH of about 4.5 and about 7.4. The amount of histidine, which is a buffer, seems to have little effect on stability, which is a stability issue for non-AS DVD-Ig proteins, as was the case with monoclonal antibodies. Is shown to be alleviated by formulation parameters.

The data show that the stability of the IL12IL18 DVD-Ig protein is maintained in the pH range of 4.5-7.4, while DVD-B is unstable in solution at both pH 4.5 and pH 7.4. Show that. However, the stability of the two DVD-Ig proteins shows a similar stability profile in the histidine concentration range from 0 to 200 mM at pH 6 under the above conditions in solution.

Example 17: Effect of citrate concentration at pH 6 on storage stability of DVD-B and IL12 IL18 DVD-Ig protein at 100 mg / mL The following example is for DVD-B and IL12 / IL18 DVD-Ig. The effect of citrate buffer concentration (range 0-100 mM) on protein storage stability is described. Samples were prepared and the storage stability of DVD-Ig protein in solution at 100 mg / mL was evaluated at 5 ° C and 40 ° C. After the prescribed storage period, samples were removed and the effect of storage time on DVD-Ig protein stability was evaluated. Briefly, samples were filled in sterile vials (approximately 500 μL each) and stored under controlled conditions (in a temperature chamber and in the absence of light) at 40 ° C. At a given point in time, a sample of the prepared solution was taken out for analysis according to the sample removal scheme.

The two DVD-Ig proteins in Table 35 showed different stability profiles between 0-100 mM citrate concentrations at pH 6. The AS-DVD-Ig protein IL12IL18 showed high stability, especially at 5 ° C., with only a small increase in aggregates over time, but the opposite for the non-AS-DVD-Ig protein DVD-B. I could say that. This indicates that high concentration liquid formulations may be feasible for the IL12IL18 DVD-Ig protein under these conditions, but not for DVD-B.

VIII. Stable lyophilized DVD-IG (LS-DVD-IG) protein formulation Examples 18 and 19 describe the stability of the LS-DVD-Ig protein in lyophilized form. Example 18 describes the surprising results demonstrating the freeze / thaw (F / T) stability of the LS-DVD-Ig protein. Example 19 describes a study showing a stable lyophilized formulation containing the LS-DVD-Ig protein. Freezing is the first step of lyophilization, so molecules that do not have lyophilization stability are prone to instability during lyophilization.

Example 18: Effect of solution pH on stability of DVD-Ig protein subjected to repeated freeze / thaw cycles DVD- at a protein concentration of 1 mg / mL in 5 mM citrate / 5 mM phosphate buffer. The freeze-thaw behavior of Ig protein was evaluated by circulating the protein solution at pH 4, pH 6, and pH 8 up to twice between the frozen and liquid states. Freezing was performed using a temperature controlled -80 ° C freezer and thawing was performed using a temperature controlled water bath at 30 ° C. Samples were removed after the second freezing / thawing (F / T) cycle and analyzed by SEC. Table 36 shows the effect of freezing / thawing processing on the amount of monomer (Mon) remaining in the samples formulated at these pH levels and the amount of fragments (Frag) and aggregates (Agg) formed. Is shown.

DVD 5, DVD 6, DVD 37, DVD 38, DVD 53, DVD 54, DVD 65, DVD 66, DVD 165, DVD 166, DVD 257, DVD 258, DVD 277, DVD 278, DVD 281 and DVD 282 Stability was demonstrated after being subjected to repeated freeze-thaw cycles. These data show that DVD-Ig proteins formulated in the pH range of about 4 to about 8 remain stable after repeated F / T processing. The high stability of the DVD-Ig protein formulations tested (all showed a monomer content of greater than 93% and 11/16 formulations showed a monomer content of greater than 95%). It was unexpected because the protein is much more complex than IgG. Complex molecules such as DVD-Ig proteins were expected to easily aggregate and fragment when exposed to freezing and thawing.

Effect of solution pH on the stability of DVD-B subjected to repeated freeze / thaw cycles Freeze-thaw behavior of DVD-B at a protein concentration of 2 mg / mL in 10 mM citrate / 10 mM phosphate buffer Was evaluated by circulating the protein solution at pH 4-9 between the frozen and liquid states up to 4 times. Freezing was performed using a temperature-controlled -80 ° C freezer, and thawing was performed using a temperature-controlled water bath at 30 ° C. Samples were removed after each freezing / thawing (F / T) cycle and analyzed by photoblocking and SEC. Table 37 shows the effect of freezing / thawing processing on the number of invisible particles formed, as determined using light blocking measurements at various pH values.

The results of the light blocking assay show that the number of particles formed by the DVD-B formulation with a pH of 4-9 was low. The number of particles formed increased with increasing pH and reached a maximum around the pI (pI 8.5) of the molecule. However, with one exception, the protein solutions tested require less than 600 particles with a size of 25 microns or more per mL. The requirements of the of Pharmaceuticals for Human Use (ICH) guidelines have been met.

Table 38 shows SEC measurements of DVD-B stability after freezing / thawing processing. These measurements include the percentage of monomers, aggregates, and fragments, as well as the area under the curve (AUC).

The results in Table 38 show that the DVD-Ig protein does not form significant amounts of aggregates even after 4 F / T cycles. This good F / T stability allows the DVD-Ig protein to undergo more degradation or further cold denaturation at the liquid / ice interface during multiple freeze / thaw cycles due to their higher structural complexity. It's amazing because it was expected to be vulnerable. This is a general observation of proteins, and many other DVD-Ig proteins show a significant amount of aggregation upon freezing / thawing. Since freezing is the first and essential step in the lyophilization process, the data suggest that DVD-B can be lyophilized and thus converted into stable biotherapeutic dosage forms. ..

The results in Table 37 show that the DVD-Ig protein does not form significant aggregates even after 4 F / T cycles. This good F / T stability may not be as good as observed, given the intrinsic lack of stability and the increased structural complexity compared to the antibody. It's amazing because it was expected to be.

Example 19: Effect of lyophilization on the stability of DVD-Ig protein Aggregates can form during the lyophilization process and later during the storage stability of the solid protein. Aggregates formed during lyophilization are generally measured after immediate reconstitution.

The storage stability of DVD-Ig protein was evaluated over a long period of time under controlled temperature conditions. After a defined storage period, samples were removed and the effects of storage time and storage temperature on the stability of lyophilized DVD-Ig proteins were evaluated by size exclusion chromatography (SEC) and ion exchange chromatography (IEC). The following three DVD-Ig proteins were studied: DVD-B (TNF-PGE2), DVD-A (TNF-IL17), and DVD-C (IL1α-IL1β). Of these three, DVD-A and DVD-C were AS-DVD-Ig proteins. DVD-B is a non-AS-DVD-Ig protein, but because it (and LS-DVD-Ig proteins DVD-A and DVD-C) was found to be stable in lyophilized formulations. It was identified as the LS-DVD-Ig protein. The formulations were lyophilized in the solutions shown in Table 39 in formulations containing buffers, polyols, and surfactants.

Table 40 shows the monomers, aggregates, and fragments measured using SEC before storage (T0) of the lyophilized formulation or after storage for a given storage period at either 5 ° C or 40 ° C. Enter the percentage of.

The SEC data in Table 40 show that the lyophilized LS-DVD-Ig proteins remain stable over a storage period of up to 3 months as they show a high percentage of monomers and a low percentage of aggregates and fragments. Indicates that there is. After 3 months of accelerated storage at 40 ° C. and 3 hours of reconstruction time, lyophilized DVD-A showed more than 93% monomers, less than 6.4% aggregates, and only about 0.4% fragments. Had. After 4 weeks of accelerated storage at 40 ° C., the lyophilized DVD-B had more than 95% monomer and only about 3.2% aggregates and about 1.4% fragments. After 1 month of accelerated storage at 40 ° C., the lyophilized DVD-C had approximately 97% monomer, 2% aggregate, and 1% fragment. Therefore, the lyophilized formulations of DVD-A, DVD-B, and DVD-C showed long-term stability when assessed by SEC.

Table 41 below provides data on the stability of stored formulations measured using IEC. The effect of chemical stability was not significant when observed from the formation of acidic and basic species over time. Therefore, all tested molecules do not show significant chemical degradation in the lyophilized state. Storage stability of lyophilized DVD-Ig protein was evaluated at 40 ° C and 5 ° C. After a defined storage period, lyophilized samples were removed and the effect of storage time on DVD-Ig protein stability was evaluated. Samples were reconstituted with water for injection and when fully reconstituted, analyzed using SEC and IEC.

The effects of chemical stability seen above in Table 40 were not significant when observed from the formation of acidic and basic species over time.

Table 42 below provides the reconstitution times of the stored lyophilized formulations. One basic criterion for characterizing the success or failure of a lyophilized dosage form is the time it takes for a lyophilized sample to be converted to a uniform, clear liquid after reconstitution with a solvent, usually water for injection. .. Ideally, the reconstruction procedure takes less than 10 minutes. As shown in Table 41, all tested samples showed complete reconstitution after up to 5 minutes. This indicates that AS-DVD-Ig proteins (eg, DVD-Ig proteins A and C) can be formulated as stable lyophilized formulations. Moreover, the above examples show that the LS-DVD-Ig protein, which is not stable in liquid formulations, can be stabilized using lyophilization (eg, DVD-B).

IX. Characterization of DVD-IG Protein Examples 20-23 provide further characterization of a general DVD-Ig protein.

Example 20: DVD-Ig Protein Solubility Rating Using PEG 3000 Polyethylene glycol (PEG) is often used as a "crowder" that pushes the protein into a small amount of water, but this is a solvent available. This is because some are bound or occupied by clauders, typically polymers. PEG is used to assess the true solubility of proteins by utilizing trace amounts of protein material available early in development. In general, the higher the amount of PEG required to induce precipitation, the higher the expected true solubility of the protein in solution.

The next study was the addition of a small amount of aliquots of PEG solution (50 w / v%) to the stock solution (0.5 mg / mL) of protein in pH 6 buffer (5 mM citrate and 5 mM phosphate). I went using it. Table 43 shows data for various DVD-Ig proteins.

These data are typical of highly soluble proteins (ie, proteins with true solubility greater than about 100 mg / mL) in which the amount of PEG 3000 required to induce precipitation of the DVD-Ig protein is typical. Indicates that. The PEG precipitation assay is a standard assay for providing information about its solubility in antibody formulation ratings, but sufficient to predict whether a DVD-Ig protein is classified as AS or LS or even non-LS. It does not appear to indicate the complexity of the DVD-Ig protein compared to monoclonal antibodies and the difficulty of tackling formulation.

Example 21: Rating of Tertiary Structure of DVD-Ig Protein Using Near UV CD Scanning Protein structure is an important factor affecting protein stability during accelerated / long-term storage of protein liquid and lyophilized formulations. It is one of them. To assess the tertiary structure of DVD-Ig protein, 250-320 nm near UV CD scans were taken with a Jasco spectrometer at a scan rate of 50 nm / min and at a concentration of 1 mg / mL. An average of 3 scans were taken per DVD-Ig protein. The pH used in the study was 6 under 5 mM citrate and 5 mM phosphate conditions. UV CD scans of monoclonal antibodies were also obtained for comparison. The data presented in FIG. 5 shows that the DVD-Ig protein has a folding structure, as indicated by the presence of a characteristic ellipticity profile in the 250-320 nm region.

The DVD-Ig ellipticity values presented in FIG. 5 are similar to those observed for known stable monoclonal antibodies. Therefore, it is expected that both DVD-Ig protein and mAb will exhibit similar storage stability. However, the LS-DVD-Ig protein is actually less stable than mAb. Therefore, UV CD data alone cannot be used to accurately predict the stability problems observed for the LS-DVD-Ig protein.

Example 22: Assessment of secondary structure of DVD-Ig protein using ATR-FTIR Region 1600 to 1700 cm by ATR-FTIR instrument from Bruker (Tensor 27) to assess secondary structure of DVD-Ig protein. Curve fitting was performed on the quadratic derivative and area-normalized FTIR scan taken in ^-1 , and various peaks were analyzed and added to obtain the total β-sheet structure% in the molecule. In particular, peaks such as a peak at 1638 cm- ¹ showing a β-sheet sequence were considered. This is because β sheets are known to form most of the secondary structure of mAbs. Therefore, it is expected that DVD-Ig proteins derived from mAbs should also contain most of the β sheets as their secondary structure composition. Any deviation from this may indicate that the overall structural integrity of the molecule is compromised. Studies were performed in 5 mM citrate / 5 mM phosphate buffer at pH 4, 6, or 8. The concentration of DVD-Ig protein was 1 mg / mL. The total percentage of β-structure was rated for 16 DVD-Ig proteins (see Table 44).

The results presented in Table 45 show that all 16 DVD-Ig proteins studied have a folded secondary structure predominantly composed of β elements. The proportion of β-elements ranges from about 85% to about 97%, similar to that observed for standard antibodies. It is assumed that the DVD-Ig proteins are engineered molecules and have a lower percentage of β-sheets compared to mAbs (but they are still β-sheets as their predominant secondary structure). It was unexpected because it is expected to contain). These findings suggest that secondary structural composition is not a factor in the lower overall storage stability of DVD-Ig proteins compared to antibodies. The proportion of β elements ranged from about 85% to about 97%.

Example 23: Effects of Ionic Strength and pH on the Secondary Virial Coefficient of DVD-B Formulation The secondary Virial coefficient (B ₂₂ ) is a thermodynamic parameter and indicator of the attractive or repulsive interaction between proteins in solution. is there. A positive value indicates a repulsive interaction and a negative value indicates an attractive interaction. Repulsive interactions usually lead to better long-term storage. The scattered light intensity is associated with the molecular weight and B ₂₂ by the following equation.

式中、Ｋは、光学定数であり、下記によって得られる。

In the formula, K is an optical constant and is obtained by the following.

Is R _theta, excess Rayleigh ratio is a measurement of light scattered by Nachi Suwa solute, n is the solvent refractive index, dn / dc is the refractive index increment of the solute, N _A is Avogadro's number And l is the wavelength of the incident light. Since the higher-order Virial coefficient has a negative value for most diluted solutions, the following equation (Debye) was used to obtain the second-order Virial coefficient.

Scattering intensity was measured by Malvern Zetasizer Nano. The secondary virial coefficient values are all positive, indicating that the DVD-Ig protein behaves as a typical protein molecule for this calculation, at least under diluting conditions. The buffers used were acetate for pH 4.5, histidine for pH 6, and Tris for pH 8. A buffer solution having a concentration of 2 mM was used for the 1 mM ionic strength solution, and 10 mM was used for the 20 and 100 mM ionic strength solutions. The remaining ionic strength was maintained by sodium chloride. The results are shown in Tables 33 and 34. The values of the secondary virial coefficient were higher on average at pH 4.5 and pH 6.0 than at pH 8.0, so DVD-B was more at pH 4.5 and pH 6.0 than at pH 8.0. It is suggested that it will be well stored. The value of the secondary virial coefficient was also higher at lower ionic strengths, suggesting that lower ionic strengths may also be associated with the stability of the TNF-PGE2 DVD-Ig protein.

D _s is the self-diffusion coefficient of the molecule at infinite dilution. _dd is a parameter that describes the interaction between molecules in solution. A positive value of k _d indicates an intermolecular repulsion and vice versa.

As mentioned, when the signs of B ₂₂ and kD are positive and large in size, it indicates a strong repulsive interaction between the DVD-Ig proteins in solution. This is an ideal condition for long-term storage stability of DVD-Ig protein liquid formulations, as it reduces the likelihood that DVD-Ig proteins will encounter each other in solution and form aggregates. The above data suggest that low ionic strength and low pH as part of the formulation results in greater long-term DVD-Ig protein stability than formulations with high ionic strength and high pH. Moreover, ionic strength and pH, since it contributes high as B ₂₂ value, which indicates that the electrostatic repulsion is generally constitutes a substantial portion of the B ₂₂ value.

For comparison of DVD-Ig protein and antibody, a more positive _B22 value may be required for the DVD-Ig protein formulation to have a long-term stability profile similar to that of the antibody formulation. is there. This is because the DVD-Ig protein is larger than the antibody and the rigid sphere's contribution to repulsion (and B ₂₂ ) is higher for the DVD-Ig protein. Therefore, if both the DVD-Ig protein and the antibody have the same electrostatic repulsion that contributes to B ₂₂ , the B ₂₂ term for the DVD-Ig protein should reflect a similar degree of interprotein repulsion in solution. , Becomes more positive.

Example 24: Pharmacokinetic studies of DVD-Ig proteins The following examples describe pharmacokinetic studies of various DVD-Ig proteins.

Variable domain combinations and orientations affect serum stability As shown in FIG. 2, certain variable domain combinations provide a more stable DVD-Ig protein than other combinations. FIG. 2A describes serum stability for a particular DVD-Ig protein in two different domain orientations, namely "outer / inner" and "inner / outer". For example, the DVD-Ig protein TNF / SOST has about 16% high molecular weight (HMW)% in the "outer / inner" orientation, while about 75% HMW is in the opposite orientation. This domain orientation concept is described in FIG. 2B. The DVD-Ig protein in FIG. 2 contains a short / short linker combination and is a huIgG1 isotype.

In vitro pharmacokinetic studies The pharmacokinetic (PK) properties of various biotherapeutic agents were assessed after a single intravenous dose of 4 mg / kg in male Sprague-Dawley rats. Blood samples were collected over the entire 28-day study. Serum samples were analyzed using an MSD assay using anti-human Ig capture and Sulfo-Tag labeled goat anti-human IgG antibody for chemiluminescence detection. Pharmacokinetic parameters for each animal were calculated by non-compartment analysis using WinNonlin software.

FIG. 3 describes the results from a pharmacokinetic study using rats. This study examined the correlation between clearance (CL) and T1 / 2 and ultra-high molecular weight (HMW) aggregate formation in rat serum. As shown in FIG. 3, DVD-Ig proteins with less than 10% HMW aggregation in vitro can have low (less than 0.3 mL / hour / kg) clearance and long (> 10 day) half-life. taller than. Outliers included DVD 257 and DVD 258. The DLL4 / VEGF DVD-Ig protein recognized a rat target and its PK was affected by TMD. DVD 037 (VEGF / HER2, SEQ ID NOs: 34 and 35) showed poor in vitro properties but good in vivo PK. Amino acid sequences for the heavy and light chains of the PLL4 / VEGF DVD-Ig protein are provided in SEQ ID NOs: 68 and 69 of Table 65.

Example 25: Viscosity Study of DVD-Ig Protein The following example describes a viscosity study of an exemplary AS-DVD-Ig protein (DVD-A).

Viscosity was measured with an m-VROC low volume viscometer manufactured by Rheosense (Redwood, CA). The m-VROC measures viscosity from its pressure drop as the test solution flows through a rectangular slit. When the test solution is pumped through the flow path, the pressure is measured at an increasing distance from the inlet. The linear plot at the pressure vs. position of the sensor is proportional to the viscosity.

The equipment was pre-exhausted to minimize material usage and then the material was recovered. Sample measurements were taken after cleaning the equipment with air. Initial flow rates from 40 μl / min to 200 μl / min were used to obtain the required pressure difference. Pressure chamber saturation rapidly stabilized the viscosity reading and achieved stabilization with a 20 microliter sample. Once the instrument was primed with the sample, less than 5 microliters of additional sample was sufficient to obtain a stable second reading. Therefore, a total of less than 30 microliters (less than 35 microliters) of sample was sufficient to obtain readings in three series.

The viscosities of all samples were also measured with a rotary sphere viscometer manufactured by Antonio Par (X, X). 1.8 mm capillaries were used for samples with a viscosity range of 2-70 cP and 1.6 mm capillaries were used for samples with a minimum viscosity (less than 2 cP). The instrument was precalibrated and performed at any of a variety of possible angles (70 °, 50 °, and / or 40 °).

The viscosity of DVDA was determined in histidine formulations with different molar concentrations (ie 0 mM to 30 mM histidine) and pH (ie pH 4.8 to pH 8.3) in various DVD-A concentrations. The results from the measurements are provided below in Tables 47-49.

The above results in Tables 47-49 show the effects of ionic strength, pH, and protein concentration on the viscosity of the DVD-Ig protein solution. DVD-A (SEQ ID NOs: 62 and 63) is an AS-DVD-Ig protein, and the above results are responsive to pharmaceutical compositions and syringe-transmissible liquid formulations at high concentrations that would be suitable for in vivo use. It is shown that the viscosity value can be adjusted by the preparation means. Not only do DVD-Ig proteins have a much higher molecular weight of about 200 kD compared to standard monoclonal antibodies of about 150 kD, but their structure is also more complex. Therefore, it was surprising that the viscosities of mAbs and DVD-Igs were relatively similar, and even at high concentrations below about 10 cPs.

Example 26: Thermal Stability of DVD-Ig Protein The following example is a DVD-containing an example AS-DVD-Ig protein and an example LS-DVD-Ig protein compared to a monoclonal antibody such as adalimumab. The results from three different tests for testing the thermal stability of Ig protein are described.

Dynamic scanning fluorescence method The OPtim-1000 automatic high-throughput device manufactured by Avacta (York, UK) was used for the study. A 9 microliter microcubic array (MCA) was used for the study. For the preparation of the sample stock solution, 3 microliters of Cypro orange (Invitrogen, Cambridge, MA) was added to 27 microliters of the sample solution to obtain a 1x final concentration of the dye. This dye is available as a 5000x commercial product, but any dye would be suitable. Thermal scanning was taken at 26 ° C. to 95 ° C. and a scanning rate of 60 ° C./hour. The baseline was fitted for linearity and the first point (temperature) at which its incorporation reduced R ² to less than 0.95 was taken as the starting temperature. Repeated studies confirmed that the variation in starting temperature was less than 5%.

Intrinsic fluorescence Tryptophan fluorescence was used to assess unfolding temperature. A Hitachi FL-4500 device manufactured by Hitachi, Japan was used for research. The temperature was maintained using a water bath. The temperature in the cuvette was measured by thermocouple and Omega Inc. It was monitored using a temperature monitor CSi32 manufactured by (Stamford, CT). A VWR (MA) front triangular crystal cuvette was used because it minimizes the Inner filter effect and thus provides a strong luminescent signal. An excitation wavelength of 295 nm was used. Luminescence was monitored at 328-338 nm. The _maximum λ _value was observed at 332 nm, while the intensity was observed at 335 nm for comparison. Thermal scanning was taken at 30 ° C. to 70 ° C. and a scanning speed of 78 ° C./hour. The baseline was fitted for linearity and the first point (temperature) at which its incorporation reduced R ² to less than 0.95 was taken as the starting temperature. Repeated studies confirmed that the variation in starting temperature was less than 5%. The increase in scanning speed did not significantly affect the starting temperature.

Differential scanning calorimetry (DSC)
DSC was used to characterize the thermodynamic stability of the protein under various solution conditions. An automatic cap (cap) manufactured by Microcal (Northampton, MA) was used. Thermal scanning was taken at 25 ° C. to 65 ° C. and a scanning rate of 60 ° C./hour. Since aggregation and precipitation following unfolding in the high concentration sample can lead to blockage of the cap DSC cell, which in turn makes cleaning considerably more difficult, scanning to prevent any such event from starting temperature. Obtained only up to approximately 5 ° C. A 10 minute pre-scan equilibration thermostat was applied prior to each scan. A corresponding buffer scan was taken immediately after the sample scan. The starting difference was less than 2 ° C. between repeated scans. The baseline was fitted for linearity and the first point (temperature) at which its incorporation reduced R ² to less than 0.95 was taken as the starting temperature. Repeated studies confirmed that the variation in starting temperature was less than 5%.

The results from this study are provided in Table 50.

The results listed in Table 50 show the effect of protein concentration on the thermal stability of the protein solution. DVD1 (IL12IL18), AS DVD and DVD2 (also referred to herein as DVD-B), LS-DVD-Ig protein, and other mAbs all have a protein concentration that is slightly relative to the thermal stability of the protein. Show that it only has an effect. Thus, the success or failure of a high-concentration liquid formulation can be independent of the effect of protein concentration on the thermal stability of the protein, but high-concentration liquid formulations exhibit other well-known types of instability, such as storage instability. In general, DVD-Ig proteins have a lower melting temperature than antibodies, as described in Examples 1-3. Similar melting temperatures are observed in some examples, such as DVD1, but in general this is not the case.

X. Anti-DLL4 / Anti-VEGF DVD-IG Formulation (Aqueous and Lyophilized)

Example 27: Pre-formulation characterization of anti-DLL4 / anti-VEGF DVD-Ig protein h1A11.1SL-Av Storage stability (5 ° C.) of anti-DLL4 / anti-VEGF DVD (h1A11.1-SL-Av, Table 41) and Accelerated stability (40 ° C.) was evaluated in the formulations listed below and at protein concentrations. Stability was evaluated by size exclusion chromatography (SEC) and the recovered aggregate%, monomer%, fragment%, and total species were quantified. Overall, the formulation covers a pH range of 5-7 and a protein concentration range of 1.0-118 mg / mL.

At temperatures of 5 ° C and 40 ° C, and at protein concentrations of 50, 30, and 10 mg / mL, the formulations were: 15 mM acetate (pH 5); 15 mM phosphate (pH 7); 30 mM at pH 5. Acetate, 80 mg / mL protein, 0.02% Tween 80; 30 mM histidine at pH 6, 80 mg / mL protein, 0.02% Tween 80; PBS (phosphate buffered saline). All formulations contained 0.02% sodium azide to inhibit microbial growth during storage. At temperatures of 5 ° C and 40 ° C, and at protein concentrations of 60, 50, 30, and 10 mg / mL, the formulation was 15 mM histidine (pH 6) (0.02 to inhibit microbial growth during storage). Also contains% sodium azide). At 5 ° C. and at a protein concentration of 118 mg / mL, the formulation was 15 mM histidine (pH 6) (also containing 0.02% sodium azide to inhibit microbial growth during storage). At 40 ° C. and at a protein concentration of 1.0 mg / mL, the formulations were 10 mM citrate and 10 mM phosphate at pH 5, 6, and 7. Formulations containing the protein were filtered to remove potential microorganisms.

Freezing-thaw stability was performed by subjecting the proteins in the formulation to freezing at -80 ° C for at least 20 hours and thawing in a water bath at 30 ° C for 4 cycles. Formulations tested for freeze-thaw stability are listed below. Stability was evaluated by SE-HPLC and the recovered aggregate%, monomer%, fragment%, and total species were quantified. The formulation is 15 mM histidine (pH 6) at 60 mg / mL protein (also contains 0.02% sodium azide to inhibit microbial growth) and 10 mM citrate and 10 mM phosphate at pH 5, 6, and 7. It was salt, as well as 1.0 mg / mL protein (filtered to remove potential microorganisms).

Finally, differential scanning calorimetry to measure thermal stability was performed on proteins in 10 mM citrate and 10 mM phosphate buffers at pH 5, 6, and 7, and 1.0 mg / mL protein. It was. The starting temperature of unfolding and the midpoint temperature (Tm) of unfolding of each protein domain were quantified.

緩衝液の解（全ての緩衝液が微生物成長を阻害するために０．０２％アジ化ナトリウムを含有する）：
酢酸塩＝１５ｍＭ酢酸塩（ｐＨ５）、ヒスチジン＝１５ｍＭヒスチジン（ｐＨ６）、リン酸塩＝１５ｍＭリン酸塩（ｐＨ７）
酢酸塩−スクロース−Ｔｗ＝３０ｍＭ酢酸塩、８０ｍｇ／ｍＬスクロース、０．０２％ Ｔｗ８０
ヒスチジン−スクロース−Ｔｗ＝３０ｍＭヒスチジン、８０ｍｇ／ｍＬスクロース、０．０２％ Ｔｗ８０
ＰＢＳ＝リン酸緩衝生理食塩水

Buffer solution (all buffers contain 0.02% sodium azide to inhibit microbial growth):
Acetate = 15 mM acetate (pH 5), histidine = 15 mM histidine (pH 6), phosphate = 15 mM phosphate (pH 7)
Acetate-sucrose-Tw = 30 mM acetate, 80 mg / mL sucrose, 0.02% Tw80
Histidine-sucrose-Tw = 30 mM histidine, 80 mg / mL sucrose, 0.02% Tw80
PBS = Phosphate Buffered Saline

解：
ＦＴ＝凍結融解
ＦＴ２＝２周期の凍結および融解、すなわち−８０℃での凍結および３０℃の水浴中での融解後の分析
ＦＴ４＝４周期の凍結および融解、すなわち−８０℃での凍結および３０℃の水浴中での融解後の分析
クエン酸塩−リン酸塩＝１０ｍＭクエン酸塩および１０ｍＭリン酸塩
ヒスチジン＝１５ｍＭヒスチジンおよび０．０２％アジ化ナトリウム（アジ化物は微生物成長を阻害するためのものである）

Solution:
FT = freeze-thaw FT2 = 2 cycles of freezing and thawing, ie freezing and thawing at -80 ° C and analysis after thawing in a water bath at 30 ° C FT4 = 4 cycles of freezing and thawing, ie freezing and thawing at -80 ° C and 30 Post-thaw analysis in a water bath at ° C Citrate-phosphate = 10 mM citrate and 10 mM phosphate histidine = 15 mM histidine and 0.02% sodium azide (azidates for inhibiting microbial growth Is a thing)

Overall, the data in Tables 51-54 suggest that h1A11.1-SL-Av has the degradation profile typically observed for stable monoclonal antibodies. Aggregation and fragmentation are higher at elevated temperatures (40 ° C) than at lower temperatures (5 ° C).

There is no apparent increase in aggregation or fragmentation after 21 days of storage at 5 ° C. At 40 ° C., the amount of degradation was higher than those values typically observed for stable mAbs. However, it was not large enough to exclude it from further development. Overall, this DVD-Ig protein was qualified as an AS-DVD-Ig protein based on its stability.

Example 28: Formulation Selection for Anti-DLL4 / Anti-VEGF DVD-Ig Protein

Materials and methods . The stability of the anti-DLL4 / anti-VEGF DVD-Ig h1A11.1-SL-Av protein was evaluated among the five formulations listed in Table 55. All formulations were prepared in 15 mM histidine buffer. Formulations F1-F4 were prepared at a protein concentration of 50 mg / mL. In these formulations, the pH is in the range of 5.5 to 6.0, the polysorbate 80 concentration is in the range of 0-0.05 w / v%, and the sucrose concentration is in the range of 0-7.5 w / v%. The arginine concentration was in the range of 0 to 1 w / v%. Formulation F4 was prepared in 15 mM histidine buffer at pH 6.0, without any stabilizers, and served as a study control for a 50 mg / mL liquid formulation stability assessment. In addition, one formulation was prepared at a protein concentration of 25 mg / mL at pH 6.0 (formulation F5). In this preparation, the polysorbate 80 concentration was 0.025 w / v% and the sucrose concentration was 3.8 w / v%.

In the above formulations, a 15 mM histidine buffer was selected because it provides sufficient buffering capacity to maintain the pH of the target formulation. Sucrose was evaluated as a stabilizer for freeze-thaw stress (lyophilization) and a stabilizer for lyophilization process-induced stress (lyophilization protection). Polysorbate 80 (surfactant) and arginine were added to potentially stabilize the formulation against aggregates and microparticle formation.

Freeze-thaw and liquid formulation stability tests . The stability of all liquid formulations was evaluated after 3 cycles of freezing / thawing (F / T) stress and after 1 month storage at -80, 5, 25, and 40 ° C. Stability by visual appearance,% aggregate% by size exclusion chromatography (SE-HPLC), charge heterogeneity by cation exchange chromatography (CEX-HPLC), reduced SDS-capillary electrophoresis (CE-SDS) It was tested by an extensive set of analytical assays, including fragmentation, invisible particles by microflow imaging (MFI), and ability to bind PLL4 / VEGF using ELISA.

Freezing / thawing and liquid stability test results are provided in Table 56.

解．ＥＦＶＰ：可視的な粒子が本質的にない、ＴＭＴＣ：数え切れないほど多い、ＮＰ：未実施

Solution. EFVP: essentially no visible particles, TMTC: countless numbers, NP: not implemented

As seen in the table above, freeze-thaw stress is due to visible particles and equivalents in formulation F4 formulated without any stabilizers (eg, polysorbate 80, sucrose, and arginine). It resulted in a high invisible particle count. Compared to other formulations, this formulation also showed a tendency for higher invisible particle numbers after 1 month storage at 25 and 40 ° C. Formulation F5 with a protein concentration of 25 mg / mL showed significantly lower aggregation compared to the 50 mg / mL formulation over 1 month storage at 5, 25, and 40 ° C.

Lyophilized formulation stability test . The stability of the selective formulation was also evaluated after the formulation was lyophilized. Lyophilized drug stability was rated for all sucrose-containing formulations (F1, F2, F3, and F5). Stability was rated after 2 weeks of storage at 55 ° C. Stability, visual appearance (before and after reconstruction), reconstruction time,% aggregate% by size exclusion chromatography (SE-HPLC), charge heterogeneity by cation exchange chromatography (CEX-HPLC), reduction Tested by an extensive set of analytical assays, including fragmentation by SDS-capillary electrophoresis (CE-SDS), invisible particles by microflow imaging (MFI), and water content by Karl Fischer titration.

The lyophilized formulation stability test results are provided in Table 57. The reconstitution time for all evaluated formulations was approximately 1 minute. A slight increase in aggregation by SEC and% basic region by CEX were observed for all formulations under stressed storage conditions at 55 ° C. Minimal changes were observed in all other measured product stability attributes.

解．ＥＦＶＰ＝可視的な粒子が本質的にない、ＮＰ＝未実施

Solution. EFVP = essentially no visible particles, NP = not implemented

The sequences of anti-DLL4 / anti-VEGF DVD-Ig protein H1A11.1-SL-Av are listed in Table 58 (DVD-Ig protein is described in Examples 28 and 29).

Table 58 provides full long and light chain sequences for DVD h1A11.1-SL-Av. The linker sequences are underlined, while the CDR and constant region sequences are in bold.

Example 29: Evaluation of pre-expansion formulation characteristics of the second anti-DLL4 / anti-VEGF DVD-Ig protein (h1A11.1-LS-Av) Different formulations are evaluated for pre-expansion formulation characteristics of the anti-DLL4 / -antiVEGF DVD-Ig protein. It was done to explore how the conditions affect the stability of the DVD-Ig protein. Data for h1A11.1-LS-Av are presented in Tables 59 and 60. Storage stability (5 ° C.) and accelerated stability (40 ° C.) of DVD-Ig proteins were evaluated in the formulations listed below and at protein concentrations. Stability was assessed by SEC and the recovered aggregate%, monomer%, fragment%, and total species were quantified. Overall, the formulation covers a pH range of 5-7 and a protein concentration range of 10-50 mg / mL.

The following formulations were evaluated at temperatures of 5 ° C and 40 ° C and at concentrations of 50, 30, and 10 mg / mL: 15 mM acetate (pH 5), 15 mM histidine (pH 6), 15 mM phosphate (pH 7),. 30 mM acetate at pH 5, 80 mg / mL sucrose, 0.02% Tween 80, 30 mM histidine at pH 6, 80 mg / mL sucrose, 0.02% Tween 80, and PBS (phosphate buffered saline). All formulations contained 0.02% sodium azide to inhibit microbial growth during storage.

緩衝液の解（全ての緩衝液が微生物成長を阻害するために０．０２％アジ化ナトリウム０．０２％を含有する）：酢酸塩＝１５ｍＭ酢酸塩（ｐＨ５）；ヒスチジン＝１５ｍＭヒスチジン（ｐＨ６）；リン酸塩＝１５ｍＭリン酸塩（ｐＨ７）；酢酸塩−スクロース−Ｔｗ＝３０ｍＭ酢酸塩、８０ｍｇ／ｍＬスクロース、０．０２％ Ｔｗｅｅｎ８０；ヒスチジン−スクロース−Ｔｗ＝３０ｍＭヒスチジン、８０ｍｇ／ｍＬスクロース、０．０２％ Ｔｗｅｅｎ８０；ＰＢＳ＝リン酸緩衝生理食塩水

Buffer solution (all buffers contain 0.02% 0.02% sodium azide to inhibit microbial growth): acetate = 15 mM acetate (pH 5); histidine = 15 mM histidine (pH 6) Phosphate = 15 mM phosphate (pH 7); acetate-sculose-Tw = 30 mM acetate, 80 mg / mL sculose, 0.02% Tween80; histidine-sculose-Tw = 30 mM histidine, 80 mg / mL sculose, 0 .02% Tween80; PBS = Phosphate Buffered Saline

The buffer solution for Table 59 is the same as that in Table 60.

Example 30: Additional DLL4-VEGF DVD-Ig protein h1A11.1. A6-LS-Av and h1A11.1. Formulation Study of A6-SL-Av A pre-expansion formulation characterization of additional DLL4 / VEGF DVD-Ig protein was performed to explore how different formulation conditions affect stability. h1A11.1. A6-LS-Av and h1A11.1. Data for the A6-SL-Av PLL4 / VEGF DVD-Ig protein are presented below. These DVD-Ig proteins were found to be unstable and failed the screening criteria for being considered AS-DVD-Ig proteins.

h1A11.1. A6-LS-Av and h1A11.1. Storage stability (5 ° C) and accelerated stability (40 ° C) of A6-SL-Av were evaluated in the formulations listed below and at protein concentrations. Stability was assessed by SEC and the recovered aggregate%, monomer%, fragment%, and total species were quantified. Overall, the formulation covers a pH range of 5-7 and a protein concentration range of 10-50 mg / mL.

The following conditions were tested at temperatures of 5 ° C and 40 ° C and at 50, 30, and 10 mg / mL: 15 mM acetate (pH 5); 15 mM histidine (pH 6); 15 mM phosphate (pH 7); pH 5 30 mM acetate, 80 mg / mL sucrose, 0.02% Tween 80; 30 mM histidine at pH 6, 80 mg / mL sucrose, 0.02% Tween 80; and PBS (phosphate buffered saline). All formulations contained 0.02% sodium azide to inhibit microbial growth during storage.

Overall, the data provided in Tables 61-63 suggests that the two DVD-Ig proteins have an atypical degradation profile not observed for stable monoclonal antibodies. Aggregation rates were in fact higher at 5 ° C than at 40 ° C.

At 5 ° C., there is a rapid increase in aggregation after 21 days of storage. At 40 ° C, the amount of degradation also increased, but not at the rate observed at 5 ° C. In both cases, aggregation was concentration-dependent.

Overall, these DVD-Ig proteins have failed screening based on instability at 5 ° C. and are examples of non-AS-DVD-Ig proteins.

緩衝液の解（全ての緩衝液が微生物成長を阻害するために０．０２％アジ化ナトリウムを含有する）：
酢酸塩＝１５ｍＭ酢酸塩（ｐＨ５）、ヒスチジン＝１５ｍＭヒスチジン（ｐＨ６）、リン酸塩＝１５ｍＭリン酸塩（ｐＨ７）
酢酸塩−スクロース−Ｔｗ＝３０ｍＭ酢酸塩、８０ｍｇ／ｍＬスクロース、０．０２％ Ｔｗ８０
ヒスチジン−スクロース−Ｔｗ＝３０ｍＭヒスチジン、８０ｍｇ／ｍＬスクロース、０．０２％ Ｔｗ８０
ＰＢＳ＝リン酸緩衝生理食塩水

Buffer solution (all buffers contain 0.02% sodium azide to inhibit microbial growth):
Acetate = 15 mM acetate (pH 5), histidine = 15 mM histidine (pH 6), phosphate = 15 mM phosphate (pH 7)
Acetate-sucrose-Tw = 30 mM acetate, 80 mg / mL sucrose, 0.02% Tw80
Histidine-sucrose-Tw = 30 mM histidine, 80 mg / mL sucrose, 0.02% Tw80
PBS = Phosphate Buffered Saline

緩衝液の解（全ての緩衝液が微生物成長を阻害するために０．０２％アジ化ナトリウムを含有する）：
酢酸塩＝１５ｍＭ酢酸塩（ｐＨ５）、ヒスチジン＝１５ｍＭヒスチジン（ｐＨ６）、リン酸塩＝１５ｍＭリン酸塩（ｐＨ７）
酢酸塩−スクロース−Ｔｗ＝３０ｍＭ酢酸塩、８０ｍｇ／ｍＬスクロース、０．０２％ Ｔｗ８０
ヒスチジン−スクロース−Ｔｗ＝３０ｍＭヒスチジン、８０ｍｇ／ｍＬスクロース、０．０２％ Ｔｗ８０
ＰＢＳ＝リン酸緩衝生理食塩水

Buffer solution (all buffers contain 0.02% sodium azide to inhibit microbial growth):
Acetate = 15 mM acetate (pH 5), histidine = 15 mM histidine (pH 6), phosphate = 15 mM phosphate (pH 7)
Acetate-sucrose-Tw = 30 mM acetate, 80 mg / mL sucrose, 0.02% Tw80
Histidine-sucrose-Tw = 30 mM histidine, 80 mg / mL sucrose, 0.02% Tw80
PBS = Phosphate Buffered Saline

緩衝液の解（全ての緩衝液が微生物成長を阻害するために０．０２％アジ化ナトリウムを含有する）：
酢酸塩＝１５ｍＭ酢酸塩（ｐＨ５）、ヒスチジン＝１５ｍＭヒスチジン（ｐＨ６）、リン酸塩＝１５ｍＭリン酸塩（ｐＨ７）
酢酸塩−スクロース−Ｔｗ＝３０ｍＭ酢酸塩、８０ｍｇ／ｍＬスクロース、０．０２％ Ｔｗ８０
ヒスチジン−スクロース−Ｔｗ＝３０ｍＭヒスチジン、８０ｍｇ／ｍＬスクロース、０．０２％ Ｔｗ８０
ＰＢＳ＝リン酸緩衝生理食塩水

Buffer solution (all buffers contain 0.02% sodium azide to inhibit microbial growth):
Acetate = 15 mM acetate (pH 5), histidine = 15 mM histidine (pH 6), phosphate = 15 mM phosphate (pH 7)
Acetate-sucrose-Tw = 30 mM acetate, 80 mg / mL sucrose, 0.02% Tw80
Histidine-sucrose-Tw = 30 mM histidine, 80 mg / mL sucrose, 0.02% Tw80
PBS = Phosphate Buffered Saline

Overall, the data in Tables 61-64 suggest that the two DVD-Ig proteins have an atypical degradation profile that is not observed for stable monoclonal antibodies. Aggregation rates are actually higher at 5 ° C than at 40 ° C. At 5 ° C., there is a rapid increase in aggregation after 21 days of storage. At 40 ° C, the amount of degradation also increased, but not at the rate observed at 5 ° C. In both cases, aggregation was concentration-dependent. Overall, these DVD-Ig proteins will not qualify as AS-DVD-Ig proteins based on their instability at 5 ° C. and will be considered aqueous instability.

Sequences The heavy and light chain amino acid sequences for the other DVD-Ig proteins described herein are provided below in Table 65. The linker sequence is underlined, while the CDR sequence is in bold. The lower "L" represents a light chain and the lower "H" represents a heavy chain.

Reference Incorporation The content of all cited references (including references, patents, patent applications, and websites) that may be cited throughout this specification is expressly expressed herein in its entirety by reference. The same applies to the references incorporated in and cited in them. Unless otherwise indicated, the practice of this disclosure uses conventional techniques of immunology, molecular biology, and cell biology that are well known in the art.

Equivalents The disclosure may be embodied in other particular forms without departing from the spirit or essential properties of the disclosure. The embodiments described above should therefore be viewed in all respects as exemplary rather than the limitations of the present disclosure described herein. The scope of the present disclosure is therefore specified not by the description above, but rather by the appended claims, and all modifications that fall within the meaning and scope of the claims are therefore described herein. Intended to be included.

Claims (76)

An aqueous formulation comprising a stable aqueous DVD-Ig (AS-DVD-Ig) protein and a buffer having a molar concentration of about 5 to about 50 mM, said formulation being about 4.5 to about 7.5. Aqueous formulation with a pH of. The AS-DVD-Ig protein, when determined by size exclusion chromatography (SEC), is about 40 when formulated in histidine or citrate phosphate buffer at a concentration of at least about 50 mg / mL. The aqueous formulation of claim 1, characterized as a DVD-Ig protein, having a loss of less than about 15% in monomeric relative percentage after 14 days of storage at degrees Celsius. The AS-DVD-Ig protein, when determined by SEC, is formulated in histidine or citrate phosphate buffer at a concentration of at least about 50 mg / mL for 14 days at about 40 degrees Celsius. The aqueous formulation according to claim 1, characterized as a DVD-Ig protein, having a loss of less than about 10% in relative proportion of monomer after storage. The AS-DVD-Ig protein, when determined by SEC, is formulated in histidine or citrate phosphate buffer at a concentration of at least about 50 mg / mL for 14 days at about 40 degrees Celsius. The aqueous formulation according to claim 1, characterized as a DVD-Ig protein, having a loss of less than about 5% in relative monomeric percentage after storage. The aqueous formulation according to claim 1, wherein the formulation comprises about 6% or less agglutination when determined by SEC analysis. The aqueous formulation according to claim 1, wherein the formulation comprises about 5% or less agglutination when determined by SEC analysis. The aqueous preparation according to any one of claims 1 to 6, wherein the preparation has a pH of about 5 to about 6.5. The AS-DVD-Ig protein is in an aqueous formulation of about 5.5 to about 6.5 pH at a concentration of about 100 mg / mL, and after storage at about 40 ° C. for 21 days, about 10% or less of the monomer relative. (Rel.) The aqueous preparation according to any one of claims 1 to 7, characterized as a DVD-Ig protein having a change in peak area. The AS-DVD-Ig protein is in an aqueous formulation of about 5.5 to about 6.5 pH, at a concentration of about 100 mg / mL, and after storage at about 5 ° C. for 21 days, about 1% or less of monomer relative. (Rel.) The aqueous preparation according to any one of claims 1 to 8, characterized as a DVD-Ig protein having a change in peak area. The aqueous preparation according to any one of claims 1 to 9, further comprising a surfactant, a polyol, and at least one component selected from a combination thereof. The aqueous preparation according to claim 10, wherein the polyol is selected from the group consisting of sorbitol, mannitol, and sucrose. The polyol is mannitol, and the concentration of mannitol is a group consisting of about 10 to about 100 mg / mL, about 20 to about 80, about 20 to about 70, about 30 to about 60, and about 30 to about 50 mg / mL. The aqueous preparation according to claim 11, which is selected from. The aqueous preparation according to claim 11, wherein the polyol is sorbitol. 13. The concentration of sorbitol is selected from the group consisting of about 20 to about 60 mg / mL, about 25 to about 55 mg / mL, about 30 to about 50 mg / mL, and about 35 to about 45 mg / mL, claim 13. Aqueous formulation. The aqueous preparation according to claim 11, wherein the polyol is sucrose. 15. The concentration of sucrose is selected from the group consisting of about 60 to about 100 mg / mL, about 65 to about 95 mg / mL, about 70 to about 90 mg / mL, and about 75 to about 85 mg / mL, claim 15. Aqueous formulation. The aqueous preparation according to any one of claims 10 to 16, wherein the surfactant is a polysorbate. The concentration of polysorbate is about 0.001% to about 1%, about 0.005% to about 0.05%, about 0.005% to about 0.02%, about 0.01% to about 0.05%. , And the aqueous formulation of claim 17, selected from the group consisting of about 0.1%. The aqueous preparation according to claim 17 or 18, wherein the polysorbate is polysorbate 80 or polysorbate 20. The aqueous preparation according to claim 19, wherein the polysorbate 80 or the polysorbate 20 has a concentration of about 0.005% to about 0.02%. The preparation according to any one of claims 1 to 20, wherein the buffer solution is selected from the group consisting of acetate, histidine, glycine, arginine, phosphate, and citrate. The aqueous preparation according to any one of claims 1 to 21, wherein the molar concentration of the buffer solution is in the range of 10 to 20 mM. The aqueous preparation according to any one of claims 1 to 22, wherein the AS-DVD-Ig protein has a concentration of about 1 to about 200 mg / mL. The AS-DVD-Ig protein is about 20 to about 100 mg / mL, about 1 to about 250 mg / mL, about 10 to about 230 mg / mL, about 20 to about 210 mg / mL, about 30 to about 190 mg / mL, about. The AS- selected from the group consisting of 40 to about 170 mg / mL, about 50 to about 150 mg / mL, about 60 to about 130 mg / mL, about 70 to about 110 mg / mL, and about 80 to about 105 mg / mL. The aqueous preparation according to claim 23, which has a concentration of DVD-Ig protein. An aqueous formulation comprising an aqueous stable DVD-Ig (AS-DVD-Ig) protein, a buffer having a molar concentration of about 5 to about 50 mM, a surfactant, and a polyol, wherein the formulation is about 4 An aqueous formulation having a pH of .5-about 7.5. The aqueous preparation according to claim 25, wherein the polyol is selected from the group consisting of sorbitol, mannitol, and sucrose. The aqueous preparation according to claim 26, wherein the polyol is sorbitol. The aqueous preparation according to claim 27, wherein the concentration of sorbitol is about 30 to about 50 mg / mL. The aqueous preparation according to claim 26, wherein the polyol is sucrose. The aqueous preparation of claim 29, wherein the sucrose has a concentration of about 70 to about 90 mg / mL. The aqueous preparation according to any one of claims 25 to 30, wherein the buffer solution is selected from the group consisting of acetate, histidine, glycine, arginine, phosphate, and citrate. The aqueous preparation according to any one of claims 25 to 31, wherein the molar concentration of the buffer solution is in the range of about 10 to about 20 mM. The aqueous preparation according to any one of claims 25 to 32, wherein the AS-DVD-Ig protein has a concentration of about 1 to about 200 mg / mL. The AS-DVD-Ig protein is about 20 to about 100 mg / mL, about 1 to about 250 mg / mL, about 10 to about 230 mg / mL, about 20 to about 210 mg / mL, about 30 to about 190 mg / mL, about. The AS- selected from the group consisting of 40 to about 170 mg / mL, about 50 to about 150 mg / mL, about 60 to about 130 mg / mL, about 70 to about 110 mg / mL, and about 80 to about 105 mg / mL. The aqueous preparation according to any one of claims 25 to 32, which has a concentration of DVD-Ig protein. A formulation comprising AS-DVD-Ig protein, polyol, buffer, and surfactant, said formulation having a pH of about 5 to about 7, said AS-DVD-Ig protein by SEC. When determining, when formulated in histidine or citrate phosphate buffer at a concentration of about 60 mg / mL, after 14 days of storage at about 40 degrees Celsius, about 15% in monomeric relative percentage. A formulation characterized as a DVD-Ig protein with less than a loss. 35. The formulation of claim 35, wherein the polyol is selected from the group consisting of sorbitol, mannitol, and sucrose. The preparation according to claim 35 or 36, wherein the buffer is selected from the group consisting of acetate, histidine, glycine, arginine, phosphate, and citrate. The preparation according to any one of claims 35 to 38, wherein the surfactant is a polysorbate. The preparation according to claim 38, wherein the polysorbate is polysorbate 80 or polysorbate 20. The preparation according to claim 39, wherein the polysorbate 80 or the polysorbate 20 has a concentration of about 0.005% to about 0.02%. A formulation comprising AS-DVD-Ig protein, polyol, histidine buffer, and polysorbate, said formulation having a pH of about 5 to about 7, said AS-DVD-Ig protein determined by SEC. When formulated in histidine or citrate phosphate buffer at a concentration of about 60 mg / mL, DVD-with less than about 15% aggregation after 14 days of storage at about 40 degrees Celsius. A formulation characterized as an Ig protein. The preparation according to any one of claims 35 to 41, which is an aqueous preparation. The preparation according to any one of claims 35 to 41, which is a lyophilized preparation. The AS-DVD-Ig protein contains first and second polypeptide chains, each independently containing VD1- (X1) n-VD2-C- (X2) n, in the formula.
VD1 is the first variable domain,
VD2 is the second variable domain,
C is a stationary domain,
X1 is a linker, provided that it is not CH1.
X2 is the Fc region
n is 0 or 1 and
The VD1 domain on the first and second polypeptide chains forms a first functional target binding site, and the VD2 domain on the first and second polypeptide chains is a second. The preparation according to any one of claims 1 to 43, which forms a functional target binding site. The first polypeptide chain comprises a first VD1- (X1) n-VD2-C- (X2) n and is described in the formula.
VD1 is the first heavy chain variable domain,
VD2 is the second heavy chain variable domain,
C is a heavy chain constant domain,
X1 is a linker, provided that it is not CH1.
X2 is the Fc region
n is 0 or 1 and
The second polypeptide chain comprises a second VD1- (X1) n-VD2-C- (X2) n and is described in the formula.
VD1 is the first light chain variable domain and
VD2 is the second light chain variable domain,
C is the light chain constant domain,
X1 is a linker, provided that it is not CH1.
X2 does not contain the Fc region
n is 0 or 1 and
The VD1 domain on the first and second polypeptide chains forms a first functional target binding site, and the VD2 domain on the first and second polypeptide chains is a second. The formulation according to claim 44, which forms a functional target binding site. The preparation according to claim 44 or 45, which comprises two first polypeptide chains and two second polypeptide chains, wherein the binding protein comprises four functional target binding sites. The preparation according to any one of claims 44 to 46, wherein X1 is not CL. The preparation according to any one of claims 44 to 47, wherein the AS-DVD-Ig protein comprises three CDRs from the variable light chain or heavy chain amino acid sequence set forth in SEQ ID NOs: 28-75. The preparation according to any one of claims 44 to 48, wherein the AS-DVD-Ig protein comprises a light chain or heavy chain amino acid sequence set forth in SEQ ID NOs: 28 to 75. The AS-DVD-Ig protein includes CD20 / CD80, VEGF / Her2, TNF / RANKL, TNF / DKK, CD20 / RANKL, PLL4 / PLGF, TNF / SOST, IL-9 / IgE, IL-12 / IL-18. , TNF / IL-17, TNF / PGE2, IL1α / IL1β, and the formulation according to any one of claims 44-49, having binding specificity selected from the group consisting of PLL4 / VEGF. The preparation according to any one of claims 44 to 49, wherein the AS-DVD-Ig protein has a binding specificity selected from the group consisting of IL4 / IL13, IL1α / IL1β, and TNFα / IL17. The preparation according to claim 50, wherein the AS-DVD-Ig protein has binding specificity for TNF and IL-17. The preparation according to claim 52, wherein the TNFα / IL17-specific AS-DVD-Ig protein is DVD A (SEQ ID NOs: 62 and 63). The preparation according to claim 50, wherein the AS-DVD-Ig protein has binding specificity for IL1α / IL1β. The preparation according to claim 54, wherein the IL1α / IL1β specific AS-DVD-Ig protein is DVD C (SEQ ID NOs: 66 and 67). The preparation according to claim 50, wherein the AS-DVD-Ig protein has binding specificity for IL-12 / IL-18. The preparation according to any one of claims 1 to 56, wherein the preparation is a pharmaceutical preparation. A pharmaceutical composition comprising an aqueous stable DVD-Ig (AS-DVD-Ig) protein and a buffer having a molar concentration of about 5 to about 50 mM, wherein the AS-DVD-Ig protein is determined by SEC. When formulated in histidine or citrate phosphate buffer at a concentration of about 60 mg / mL, after 14 days of storage at about 40 degrees Celsius, less than about 10% in monomeric relative percentage. A pharmaceutical composition characterized as a DVD-Ig protein with a loss of, said formulation having a pH of 4.5-7.5. The pharmaceutical composition of claim 58, further comprising a surfactant, a polyol, and at least one ingredient selected from combinations thereof. A method of treating a disorder comprising administering the pharmaceutical composition according to claim 58 or 59 so that the disorder is treated. A lyophilized formulation comprising a lyophilized stable DVD-Ig (LS-DVD-Ig) protein, wherein when the formulation is reconstituted, it is about 1 to about 100 mg / mL of the LS-DVD-. A lyophilized formulation comprising Ig protein, about 10 to about 50 mM buffer, polyol, about 0.01 to about 0.2 mg / mL polysorbate and having a pH of about 5 to about 7. The lyophilized preparation according to claim 61, wherein the preparation has a pH of about 5.5 to about 6.5. The LS-DVD-Ig protein is accelerated storage in an aqueous formulation at a pH of about 5.5 to about 6.5 at about 40 ° C. for 21 days when formulated at concentrations above about 100 mg / mL. The lyophilized formulation according to claim 61 or 62, wherein a monomer relative (rel.) Peak area change of greater than 10% is later observed. The LS-DVD-Ig protein contains first and second polypeptide chains, each independently containing VD1- (X1) n-VD2-C- (X2) n, in the formula.
VD1 is the first variable domain,
VD2 is the second variable domain,
C is a stationary domain,
X1 is a linker, provided that it is not CH1.
X2 is the Fc region
n is 0 or 1 and
The VD1 domain on the first and second polypeptide chains forms a first functional target binding site, and the VD2 domain on the first and second polypeptide chains is a second. The lyophilized preparation according to any one of claims 61 to 63, which forms a functional target binding site. The first polypeptide chain comprises a first VD1- (X1) n-VD2-C- (X2) n and is described in the formula.
VD1 is the first heavy chain variable domain,
VD2 is the second heavy chain variable domain,
C is a heavy chain constant domain,
X1 is a linker, provided that it is not CH1.
X2 is the Fc region
n is 0 or 1 and
The second polypeptide chain comprises a second VD1- (X1) n-VD2-C- (X2) n and is described in the formula.
VD1 is the first light chain variable domain and
VD2 is the second light chain variable domain,
C is the light chain constant domain,
X1 is a linker, provided that it is not CH1.
X2 does not contain the Fc region
n is 0 or 1 and
The VD1 domain on the first and second polypeptide chains forms a first functional target binding site, and the VD2 domain on the first and second polypeptide chains is a second. The lyophilized formulation of claim 64, which forms a functional target binding site. The lyophilized preparation according to claim 64 or 65, which comprises two first polypeptide chains and two second polypeptide chains, wherein the binding protein comprises four functional target binding sites. The lyophilized preparation according to any one of claims 64 to 66, wherein X1 is not CL. The lyophilization of any one of claims 64-67, wherein the LS-DVD-Ig protein comprises three CDRs from the variable light chain or heavy chain amino acid sequence set forth in SEQ ID NOs: 28-75. Formulation. The lyophilized preparation according to any one of claims 63 to 66, wherein the LS-DVD-Ig protein comprises a light chain or heavy chain amino acid sequence set forth in SEQ ID NOs: 28 to 75. The LS-DVD-Ig protein is CD20 / CD80, VEGF / Her2, TNF / RANKL, TNF / DKK, CD20 / RANKL, PLL4 / PLGF, TNF / SOST, IL-9 / IgE, IL-12 / IL-18. , TNF / IL-17, TNF / PGE2, IL1α / IL1β, or the lyophilized preparation according to any one of claims 61 to 69, which has binding specificity selected from the group consisting of DLL4 / VEGF. .. The lyophilized preparation according to claim 70, wherein the LS-DVD-Ig protein has binding specificity for TNF and IL-17. The lyophilized preparation according to claim 70, wherein the LS-DVD-Ig protein has binding specificity for IL1α / IL1β. The lyophilized preparation according to claim 70, wherein the LS-DVD-Ig protein has binding specificity for IL-12 / IL-18. A lyophilized formulation prepared by lyophilizing an aqueous formulation comprising LS-DVD-Ig protein, a buffer solution having a molar concentration of about 5 to about 50 mM, a surfactant, and a polyol. Is a lyophilized formulation having a pH of about 4.5 to about 7.5. The lyophilized preparation according to claim 74, wherein the buffer is selected from the group consisting of histidine, succinate, and citrate and / or phosphate. The lyophilized preparation according to claim 74 or 75, wherein the polyol is selected from the group consisting of mannitol, sorbitol, sucrose, and trehalose.

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